Nucleic acid comprising chimeric gene derived from hepatitis C virus

TECHNICAL FIELD

The present invention relates to a nucleic acid comprising a chimeric gene derived from hepatitis C viruses, a chimeric hepatitis C virus particle of the JFH-1 strain and a strain other than the JFH-1 strain (preferably a strain of genotype 1a, 1b, or 2a), a vector used for producing the virus particle, and a cell that produces the virus particle.

The present invention also relates to a method for screening for an anti-HCV drug using the virus particle, a vaccine obtained by inactivating or attenuating the virus particle, and an anti-hepatitis C virus antibody that recognizes the virus particle as an antigen.

BACKGROUND ART

Hepatitis C virus (which may be simply referred to as "HCV" hereinafter) was discovered and identified as a causative virus of non-A and non-B hepatitis by Choo et al. in 1989 (Choo, Q L. et al., Science, 244: 359-362, 1989). HCV infection is a major cause for the progression with persistent infection from chronic hepatitis to cirrhosis and then hepatic cancer. It is said that there are approximately 170,000,000 HCV patients in the world and approximately 2,000,000 HCV patients in Japan. The major route of infection is transmission through blood. Since the screening of blood for transfusion was made available, the number of people newly infected with HCV dramatically decreased in Japan. However, it is considered that many virus carriers still remain.

At present, HCV is mainly treated with the use of PEG-interferon or with the use of PEG-interferon in combination with an antiviral drug (i.e., ribavirin). HCV is currently classified into 6 different genotypes, and HCV of genotype 1b and of genotype 2a are major types in Japan. In the case of HCV of genotype 1b, in particular, viruses cannot be completely eliminated from the body via administration of interferon and ribavirin, and therapeutic effects are insufficient. Accordingly, development of novel antiviral drugs or vaccines aimed at preventing virus carriers from causing the disease to develop or at eliminating viruses has been awaited.

The lack of effective animals that reflect virus infection besides chimpanzees and the lack of an effective in vitro virus culture system had been impediments to the development of therapeutic agents of HCV. In recent years, HCV replicon systems that enable evaluation of HCV-RNA replication have been developed (Lohmann, V. et al., Science., 285: 110-113, 1999), and such systems resulted in important progress as a system for screening for HCV inhibitors associated with the inhibition of virus replication.

HCV is a single-stranded (+) RNA virus having a genome length of approximately 9.6 kb, which has a gene encoding a precursor protein converted into 10 types of virus proteins (i.e., Core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins) upon post-translational cleavage by proteases. The replicon system was prepared by recombining the translation region of the HCV structural protein with a drug-resistant gene and inserting IRES of the encephalomyocarditis virus (EMCV) downstream thereof. RNA replication is observed in cells into which such recombinant RNA has been introduced. Even when a full-length genome RNA having the HCV structural protein region has been introduced into cells, however, release of virus particles into the culture solution has not been observed (Pietschmann, T. et al., J. Virol., 76: 4008-4021, 2002).

Recently, the HCV JFH-1 strain of genotype 2a isolated from a patient with fulminant hepatitis was discovered by Wakita et al., and the strain was found to be released as infectious virus particles in a culture medium of Huh-7 cells (hepatic cancer cell lines) (WO 05080575A1 and Wakita, T. et al., Nat. Med., 11: 791-796, 2005). This in vitro culture system for infectious HCV particles is expected to serve as a useful screening tool in the development of anti-HCV agents and to be an effective means for preparation of HCV vaccines. Research on HCV particle production in in vitro culture systems has made progress, and the HCV genome capable of virus particle production was found to be chimeric HCV of the JFH-1 strain and an HCV strain other than the JFH-1 strain. Such chimeric HCV can be prepared by recombining the structural genes of the JFH-1 genome (i.e., Core, E1, E2 and p7 protein coding sections) with structural genes of other HCV strains.

As chimeric HCV of the JFH-1 strain and an HCV strain other than the JFH-1 strain, the chimeric HCV of the J6CF strain (genotype 2a) and the JFH-1 strain (Lindenbach, B. D. et al., Science, 309: 623-626, 2005), the chimeric HCV of the H77 strain (genotype 1a) and the JFH-1 strain (WO 06096459A2 and MinKyung, Y. et al., J. Virol., 81: 629-638, 2007), and the chimeric HCV of the S52 strain (genotype 3a) and the JFH-1 strain (Gottwein, J M et al., Gastroenterology 133: 1614-1626, 2007) are known.

Pietschmann, T. et al. (Proc. Natl. Acad. Sci. U.S.A., 103: 7408-7413, 2006) discloses that the amount of viruses produced from chimeric HCV of the J6CF structural gene and the JFH-1 non-structural gene is the highest and the amount of infectious virus particles produced from chimeric HCV of the Con1 strain of genotype 1b and the JFH-1 strain is one tenth the former amount. As other chimeric HCV of a genotype 1b strain and the JFH-1 strain, WO 06022422A1 discloses that a genome (full-length genome replicon RNA) in which a region encoding the structural proteins of the TH strain is recombined with that of the JFH-1 genome, and a drug-resistant gene is inserted into a site upstream of such coding region was produced, the resulting genome is introduced into the Huh-7 cell, and then a drug-resistant strain is obtained, and infectious virus particles are produced into the culture supernatant, although productivity thereof is not clearly described.

Under such circumstances, concerning genotype 1b for which the possibility of attaining a complete remission with current therapy techniques is small and of which the number of patients is large, there is a need for development of a method for producing HCV particles that can produce large quantities of infectious virus particles with the structure of genotype 1b, and that can be cultured in a persistent infection system.

DISCLOSURE OF THE INVENTION

Problem to Be Solved by the Invention

It is an object of the present invention to provide a method for efficiently producing HCV particles having the structural protein(s) of an HCV strain other than the JFH-1 strain of genotype 1a, 1b, or 2a and vaccines and the like comprising the resulting HCV particles.

Means for Solving the Problem

The present inventors have conducted concentrated studies in order to solve the above problem. They examined the ability to produce HCV particles via cell culture and discovered an adaptive mutation that appears during HCV proliferation. They demonstrated that introduction of such adaptive mutation yields the significantly enhanced ability to produce HCV particles compared with wild-type strains before introduction of such mutation, and that HCV particles having the structural protein of the HCV strain of genotype 1a, 1b, or 2a can be prepared in a persistent infection system. This has led to the completion of the present invention.

Specifically, the present invention relates to (1) to (22) below.

(1) A nucleic acid comprising a chimeric gene derived from hepatitis C viruses comprising regions each encoding, Core protein, E1 protein, E2 protein and p7 protein derived from a hepatitis C virus strain other than JFH-1 strain; NS2 protein derived from JFH-1 strain or a hepatitis C virus strain other than JFH-1 strain, or a chimeric NS2 protein of NS2 protein derived from JFH-1 strain and NS2 protein derived from a hepatitis C virus strain other than JFH-1 strain; and NS3 protein, NS4A protein, NS4B protein, NS5A protein and NS5B protein derived from JFH-1 strain in that order in 5' to 3' direction,

wherein the 328th proline residue from the amino acid residue at N-terminus of the Core protein is substituted with an amino acid residue other than proline.

(2) The nucleic acid according to (1) above, which comprises 5'-untranslated region of JFH-1 strain on the 5' side of the Core protein coding region and 3'-untranslated region of JFH-1 strain on the 3' side of the NS5B protein coding region.

(3) The nucleic acid according to (1) or (2) above, wherein the hepatitis C virus strain other than JFH-1 strain is of genotype 1a, 1b, or 2a.

(4) The nucleic acid according to any one of (1) to (3) above, wherein the hepatitis C virus strain other than JFH-1 strain is selected from the group consisting of TH strain, Con1 strain, J1 strain and derivative strains thereof.

(5) The nucleic acid according to any one of (1) to (4) above, wherein the amino acid residue other than proline is selected from the group consisting of Ala, Leu, Ile, Val, Thr and Ser.

(6) The nucleic acid according to any one of (1) to (5) above, wherein the nucleic acid is DNA comprising the nucleotide sequence as shown in SEQ ID NO: 1 of the sequence listing or a nucleotide sequence having 90% or more identity to the nucleotide sequence as shown in SEQ ID NO: 1, or RNA comprising the nucleotide sequence as shown in SEQ ID NO: 3 of the sequence listing or a nucleotide sequence having 90% or more identity to the nucleotide sequence as shown in SEQ ID NO: 3.

(7) The nucleic acid according to any one of (1) to (5) above, wherein the nucleic acid is DNA comprising the nucleotide sequence as shown in SEQ ID NO: 2 of the sequence listing or a nucleotide sequence having 90% or more identity to the nucleotide sequence as shown in SEQ ID NO: 2, or RNA comprising the nucleotide sequence as shown in SEQ ID NO: 4 of the sequence listing or a nucleotide sequence having 90% or more identity to the nucleotide sequence as shown in SEQ ID NO: 4.

(8) A vector comprising the nucleic acid according to any one of (1) to (7) above.

(9) A chimeric hepatitis C virus particle comprising the nucleic acid according to any one of (1) to (7) above as a virus genome.

(10) A cell which produces the chimeric hepatitis C virus particle according to (9) above.

(11) The cell according to (10) above, wherein the cell is of Huh-7 strain or a derivative strain thereof.

(12) A method for screening for an anti-hepatitis C virus substance comprising culturing, in the presence of a test substance:

(a) the cell according to (10) or (11) above; or

(b) the chimeric hepatitis C virus particle according to (9) above and a hepatitis C virus-sensitive cell, and detecting replicon RNA or a virus particle derived from above-mentioned nucleic acid in the resulting culture.

(13) A hepatitis C virus vaccine comprising the chimeric hepatitis C virus particle according to (9) above.

(14) The hepatitis C virus vaccine according to (13) above, wherein the chimeric hepatitis C virus particle is inactivated or attenuated.

(15) An anti-hepatitis C virus antibody which recognizes the chimeric hepatitis C virus particle according to (9) above as an antigen.

(16) The nucleic acid according to (4) above, wherein the hepatitis C virus strain other than JFH-1 strain is TH strain or a derivative strain thereof.

(17) The nucleic acid according to (5) above, wherein the amino acid residue other than proline is Ala or Thr.

(18) A method for producing a chimeric hepatitis C virus particle comprising steps of:

culturing the cell according to (10) or (11) above; and

recovering the chimeric hepatitis C virus particle according to (9) above.

(19) A method for producing a hepatitis C virus vaccine comprising steps of:

inactivating or attenuating the chimeric hepatitis C virus particle according to (9) above to prepare an inactivated or attenuated chimeric hepatitis C virus particle; and

formulating the inactivated or attenuated chimeric hepatitis C virus particle into a hepatitis C virus vaccine.

(20) A method for producing an anti-hepatitis C virus antibody comprising a step of immunizing an animal (excluding a human) with the chimeric hepatitis C virus particle according to (9) above that is or is not inactivated or attenuated.

(21) The method according to (20) above, wherein the anti-hepatitis C virus antibody is a polyclonal or monoclonal antibody.

(22) The method according to (20) above, wherein the anti-hepatitis C virus antibody is a humanized antibody.

Effects of the Invention

The nucleic acid comprising a chimeric gene derived from hepatitis C viruses of the present invention can be used for producing chimeric HCV particles exhibiting significantly higher productivity than wild-type HCV particles. The chimeric HCV particles of the present invention are advantageous over wild-type HCV particles in terms of their significantly high ability to be produced and their high infectivity with cells. Thus, the utility value thereof is high for a vaccine for HCV prevention or treatment or as a tool used for inducing an antibody reacting with HCV.

This description includes the contents of the description and/or drawings of Japanese Patent Application No. 2008-116193, which is a priority document of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a method for preparing the pTH/JFH1 plasmid. In the pJFH1 sequence, a nucleotide sequence encoding amino acid residues 1 to 846 of the amino acid sequence of the JFH-1 Core protein when the amino acid residue at the N-terminus of the JFH-1 is designated as amino acid residue 1 is substituted with a nucleotide sequence encoding amino acid residues 1 to 843 of the amino acid sequence of the Core protein of the TH strain when the amino acid residue at the N terminus of the TH strain is designated as amino acid 1.

FIG. 2 shows changes in concentrations of the HCV Core protein in the culture supernatant assayed during each passage in repetitive subcultures conducted after introducing RNA synthesized from pTH/JFH1 into the Huh-7 cell. The amount of the Core protein in the culture supernatant continued to decrease until day 23, and it increased thereafter and maintained a constantly high level after day 34.

FIG. 3A shows a method for preparing the pTH/JFH1(PA) plasmid.

FIG. 3B shows a method for preparing the pTH/JFH1(PT) plasmid.

FIG. 4 shows changes in concentrations of the HCV Core protein in the culture supernatant assayed during each passage in repetitive subcultures conducted after introducing RNAs synthesized from pTH/JFH1, pTH/JFH1(PA) and pTH/JFH1(PT) into the Huh-7 cells. As with the case shown in FIG. 2, the Core protein amount of TH/JFH-1 continued to decrease until 29 days after introduction but it increased thereafter. In the case of the cells into which TH/JFH-1 (PA) and TH/JFH-1 (PT) had been introduced, however, a large quantity of Core protein was observed in the culture supernatant from the initial stage after the introduction, and the quantities of both core proteins were maintained at higher levels than that of TH/JFH-1.

FIG. 5A shows changes in concentrations of the HCV Core protein in the culture supernatant until 96 hours had passed since RNAs synthesized from pTH/JFH1 and pTH/JFH1(PA) had been introduced into the Huh-7 cells. The amount of Core protein produced in the culture supernatant of TH/JFH-1(PA) became greater 48 hours after RNA introduction and later, than that of TH/JFH-1.

FIG. 5B shows the infectious titer attained when uninfected Huh-7 cells are inoculated with the culture supernatants obtained in FIG. 5A. The infectious titer in the culture supernatant of TH/JFH-1(PA) became greater 48 hours after RNA introduction and later, than that of TH/JFH-1.

The table shown in FIG. 6 shows the results of quantification of concentrations of HCV Core protein in the culture supernatants obtained by the experiments shown in FIG. 5, the infectious titers for the Huh-7 cells, and concentrations of HCV Core protein in the cells. The proportion of Core protein after 96 hours relative to that of Core proteins in the cells after 4 hours represents the degree of autonomous replication of RNAs of TH/JFH-1 and of TH/JFH-1(PA) in the cells. The efficiency of HCV Core secretion serves as an indicator of the efficiency of HCV particle secretion in the culture supernatant. Such efficiency was calculated by dividing the amount of Core protein in the culture supernatant after 96 hours by the sum of the amount of Core protein in the culture supernatant after 96 hours and the amount of Core protein in the cell after 96 hours. The efficiency of HCV particle secretion in the culture supernatant in the cells into which RNA of TH/JFH-1(PA) had been introduced was higher than that of TH/JFH-1.

BEST MODES FOR CARRYING OUT THE INVENTION

In general, the nucleic acid of the present invention comprises a chimeric gene of HCV comprising a nucleotide sequence encoding non-structural proteins of the JFH-1 strain and a nucleotide sequence encoding structural proteins of an HCV strain other than the JFH-1 strain. In addition, the nucleic acid of the present invention comprises a nucleotide sequence encoding the E1 protein having a given amino acid mutation.

Specifically, the nucleic acid of the present invention comprises:

(1) a chimeric gene derived from the hepatitis C viruses comprising regions each encoding the Core protein, the E1 protein, the E2 protein and the p7 protein derived from a hepatitis C virus strain other than the JFH-1 strain, the NS2 protein derived from the JFH-1 strain or a hepatitis C virus strain other than the JFH-1 strain, or the chimeric NS2 protein of the NS2 protein derived from the JFH-1 strain and the NS2 protein derived from a hepatitis C virus strain other than the JFH-1 strain, and the NS3 protein, the NS4A protein, the NS4B protein, the NS5A protein, and the NS5B protein derived from the JFH-1 strain in that order in the 5' to 3' direction; and

(2) a nucleotide sequence encoding a combined protein (i.e., a precursor protein) in which the amino acid residue 328 when an amino acid residue at the N terminus of the Core protein; i.e., a methionine residue, is designated as amino acid residue 1 (or amino acid residue 137 when the amino acid residue at the N terminus of the E1 protein is designated as amino acid residue 1); i.e., the proline residue is substituted with an amino acid residue other than proline.

The NS2 protein of the nucleic acid of the present invention may be derived from the JFH-1 strain, it may be derived from an HCV strain other than the JFH-1 strain, and it may be a chimeric protein comprising part of the NS2 protein derived from an HCV strain other than the JFH-1 strain and remaining part of the NS2 protein derived from the JFH-1 strain. In such a case, such chimeric protein has functions similar to those of the wild-type NS2 protein. When part of the NS2 protein derived from an HCV strain other than the JFH-1 strain is composed of an amino acid sequence from the N-terminus to amino acid 33 of the NS2 protein, for example, the remaining part of the NS2 protein derived from the JFH-1 strain consists of an amino acid sequence from amino acid 34 to the C terminus.

Examples of the chimeric gene derived from hepatitis C viruses include DNA consisting of nucleotides 341 to 9433 of SEQ ID NO: 1 or 2 of the sequence listing and RNA consisting of nucleotides 341 to 9433 of SEQ ID NO: 3 or 4 of the sequence listing.

According to an embodiment of the present invention, the nucleic acid of the present invention can further comprise the 5'-untranslated region of the JFH-1 strain on the 5' side of the Core protein coding region and the 3'-untranslated region of the JFH-1 strain on the 3' side of the NS5B protein coding region.

According to an embodiment of the present invention, the HCV strain other than the JFH-1 strain is of genotype 1a, 1b, or 2a. Examples of strains of genotype 1b include the TH strain, the Con1 strain, the J1 strain and derivative strains thereof. An example of a strain of genotype 1a is the H77 strain. An example of a strain of genotype 2a is the J6CF strain. Examples of preferable strains are those of genotype 1b as exemplified above. The TH strain or a derivative strain thereof is more preferable. In the present invention, the 328th amino acid residue from the amino acid residue at the N terminus of the Core protein derived from the exemplified strain must be mutated into an amino acid residue other than proline.

According to another embodiment of the present invention, the amino acid residue other than proline is, for example, Ala, Leu, Ile, Val, Thr, or Ser, and it is preferably Ala or Thr.

According to an embodiment of the present invention, the nucleic acid is DNA comprising the nucleotide sequence as shown in SEQ ID NO: 1 or RNA comprising the nucleotide sequence as shown in SEQ ID NO: 3. According to another embodiment, the nucleic acid is DNA comprising the nucleotide sequence as shown in SEQ ID NO: 2 or RNA comprising the nucleotide sequence as shown in SEQ ID NO: 4. Such nucleic acids are chimeric nucleic acids derived from the JFH-1 strain and the TH strain. The nucleic acid as shown in SEQ ID NO: 1 or 3 has a nucleotide sequence identical to that of the nucleic acid as shown in SEQ ID NO: 2 or 4, except that the former sequence comprises a codon (nucleotides 1322 to 1324) encoding Ala as the 328th amino acid residue from the amino acid residue at the N terminus of the Core protein while the nucleic acid as shown in SEQ ID NO: 2 or 4 comprises a codon (nucleotides 1322 to 1324) encoding Thr as the 328th amino acid residue from the amino acid residue at the N terminus of the Core protein.

Further, the amino acid sequence encoded by the nucleotide sequence corresponding to ORF of DNA as shown in SEQ ID NO: 1 (i.e., the sequence from the N terminus of Core to the C terminus of NS5B) is shown in SEQ ID NO: 6. The amino acid sequence encoded by the nucleotide sequence corresponding to ORF of DNA as shown in SEQ ID NO: 2 (i.e., the sequence from the N terminus of Core to the C terminus of NS5B) is shown in SEQ ID NO: 7.

The nucleotide sequence of the present invention may comprise a nucleotide sequence having 90% or more, preferably 95% or more, and more preferably 98% to 99% or more identity to the nucleotide sequence as shown in SEQ ID NO: 1, 2, 3, or 4. In such a case, the 1,322nd to 1,324th residues from the 5' terminus of the nucleotide sequence as shown in SEQ ID NO: 1, 2, 3, or 4 encode amino acid residues other than proline (see above).

The amino acid sequence of the present invention may comprise an amino acid sequence having 90% or more, preferably 95% or more, and more preferably 98% to 99% or more identity to the amino acid sequence as shown in SEQ ID NO: 5 or 6. In such a case, the 328th residue from the amino acid residue at the N terminus of the Core protein encodes an amino acid residue other than proline (see above).

This is because, as several genotypes of HCV, which is an RNA virus, are known, the structural region, the non-structural region, and/or the (5'- or 3'-) untranslated region of HCV are likely to undergo mutation.

In the present invention, the term "the 328th amino acid residue from the amino acid residue at the N terminus of the Core protein" used above refers to an amino acid residue that is aligned at the same position as that of the 328th amino acid residue of the sequence as shown in SEQ ID NO: 5, 6, or 7 in the amino acid sequence comprising a region from the N terminus of HCV Core to the C terminus of NS5B that is aligned with the amino acid sequence as shown in SEQ ID NO: 5, 6, or 7 of the sequence listing (i.e., the sequence from the N terminus of Core to the C terminus of NS5B). Also, the condition in which "the 328th amino acid residue from the amino acid residue at the N terminus of the Core protein derived from the exemplified strain is substituted with an amino acid residue other than proline" is a condition in which an amino acid residue in a given amino acid sequence that is aligned at the same position as that of the 328th amino acid residue of the sequence as shown in SEQ ID NO: 5 is an amino acid residue other than proline.

The term "% identity" used with reference to two sequences herein refers to a function of the number of positions that two nucleotide or amino acid sequences share, and it refers to a percentage of the number of matched positions relative to the total number of positions when two sequences are aligned with or without the introduction of a gap. % identity can be determined with the use of mathematical algorithms, such as BLASTN, BLASTX, or Gapped BLAST (e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A., 90: 5873-5877, 1993; Altschul et al., Nucleic Acids Res., 25: 3389-3402, 1997).

The present invention provides a vector or a chimeric HCV particle comprising the above nucleic acid. Such chimeric HCV particle can be produced with higher efficiency in a cell culture system, compared with a wild-type particle, and it has higher infectivity. Advantages such as high-efficiency production or high infectivity result from mutation of the 328th amino acid residue from the amino acid residue at the N terminus of the Core protein with an amino acid residue other than Pro (preferably Ala or Thr), which are apparent from the results shown in FIGS. 4 and 5.

The nucleic acid, the vector, and the chimeric HCV particle of the present invention can be prepared via conventional molecular biological, virological, or other techniques within the scope of the technology of the relevant field. Such techniques are described in academic documents, patent documents, specialized books, and the like. Examples of such documents include Sambrook et al., Molecular Cloning: A Laboratory Manual (vol. 3, 2001, CSHL PRESS), Mahy et al., Virology: a practical approach (1985, IRL PRESS), Ausubel et al., Current Protocols in Molecular Biology (vol. 3, 1995, John Wiley & Sons), and U.S. Pat. No. 4,683,202 (Cetus Corporation; PCR).

In order to produce the nucleic acid or infectious HCV particles of the present invention, a polymerase chain reaction (PCR) is carried out with the use of a vector prepared via cloning of cDNA of genome RNA derived from the JFH-1 strain or an HCV strain other than the JFH-1 strain as a template and the forward and the reverse primers designed based on the sequence of the cDNA, so that the target sequence portion can be amplified. As shown in FIG. 1 or 3, specifically, a plurality of different PCR products having overlapping sequences are synthesized, such PCR products are mixed, the resultant is used as a template, and PCR is carried out using a forward primer comprising the 5' end of the target nucleic acid and a reverse primer comprising the 5' end of a complementary strand of such nucleic acid, so that the target nucleic acid can be amplified. The ends of the synthesized nucleic acids are cleaved with restriction enzymes and ligated to the pJFH1 plasmid that was cleaved with the same enzyme (Wakita, T. et al., Nat. Med., 11: 791-796, 2005; WO 2004/104198). The basic technique of such procedure is also described in, for example, WO 04104198 A1, WO 06022422 A1, Wakita, T. et al., Nat. Med., 11: 791-796, 2005, and Lindenbach, B. D. et al., Science, 309: 623-626, 2005.

PCR is carried out in the presence of a template, primers, dNTPs, heat-resistant polymerase, and Mg.sup.2+-containing buffer, and a step of 94.degree. C. to 98.degree. C. for about 10 to 60 seconds, 55.degree. C. to 58.degree. C. for about 10 to 60 seconds, and 72.degree. C. for about 30 to 60 seconds is repeated 20 to 40 times, although PCR conditions are not limited thereto.

In general, the HCV genome is RNA comprising the 5'-untranslated region, the Core protein coding region, the E1 protein coding region, the E2 protein coding region, the p7 protein coding region, the NS2 protein coding region, the NS3 protein coding region, the NS4A protein coding region, the NS4B protein coding region, the NS5A protein coding region, the NS5B protein coding region, and the 3'-untranslated region. In contrast, the nucleic acid of the present invention capable of producing infectious HCV particles is composed of virus genome RNA of two or more types of HCV strains or DNA encoding such RNA.

The nucleic acid of the present invention comprises, for example, the 5'-untranslated region, the region encoding part of the NS2 protein, the NS3 protein coding region, the NS4A protein coding region, the NS4B protein coding region, the NS5A protein coding region, the NS5B protein coding region, and the 3'-untranslated region, which are derived from the JFH-1 strain, and the Core protein coding region, the E1 protein coding region, the E2 protein coding region, the p7 protein coding region, and the region encoding remaining part of the NS2 protein, which are derived from an HCV strain other than the JFH-1 strain. The 5' untranslated region may be derived from an HCV strain other than the JFH-1 strain.

According to another embodiment of the present invention, the chimeric nucleic acid of the present invention comprises the 5'-untranslated region, the Core protein coding region, the E1 protein coding region, the E2 protein coding region, the p7 protein coding region and the NS2 protein coding region, which are derived from an HCV strain other than the JFH-1 strain and the NS3 protein coding region, the NS4A protein coding region, the NS4B protein coding region, the NS5A protein coding region, the NS5B protein coding region and the 3'-untranslated region, which are derived from the JFH-1 strain.

According to another embodiment of the present invention, the nucleic acid of the present invention comprises the 5'-untranslated region derived from the JFH-1 strain; the Core protein coding region, the E1 protein coding region, the E2 protein coding region, the p7 protein coding region and the region encoding part of the NS2 protein derived from the TH strain; and the region encoding another part of the NS2 protein, NS3 protein coding region, the NS4A protein coding region, the NS4B protein coding region, the NS5A protein coding region, the NS5B protein coding region, and the 3'-untranslated region derived from the JFH-1 strain. As long as the Core protein coding region, the E1 protein coding region, the E2 protein coding region, and the p7 protein coding region are derived from the TH strain, however, the chimeric nucleic acid is not limited thereto.

Hereafter, use of the vector, the infectious HCV particles, HCV particle-producing cells, and HCV particles of the present invention are described in greater detail.

(1) Vector Preparation

The hepatitis C virus (HCV) genome is a single-stranded (+) RNA comprising approximately 9,600 nucleotides. This genomic RNA comprises a 5'-untranslated region (also referred to as "5' NTR" or "5' UTR"), a translational region composed of structural regions and non-structural regions, and a 3'-untranslated region (also referred to as "3' NTR" or "3' UTR"). In the structural regions, HCV structural proteins are encoded and, in the non-structural regions, a plurality of non-structural proteins are encoded.

Such HCV structural proteins (Core, E1, E2 and p7) and non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) are first translated as a continuous polyprotein (i.e., precursor protein) from the translational region, subjected to limited degradation by protease in infected cells, and released and produced. Among such structural and non-structural proteins (i.e., HCV virus proteins), Core is a core protein, and E1 and E2 are envelope proteins. It is known that non-structural proteins are associated with replication of the virus itself, NS2 has metalloprotease activity, and NS3 has serine protease activity (one-third of the N-terminal side) and helicase activity (two-thirds of the C-terminal side). Further, it is reported that NS4A is a cofactor for protease activity of NS3, and NS5B has RNA-dependent RNA polymerase activity.

HCV is coated with a coat referred to as an envelope. The envelope comprises a component derived from the membrane of a host cell and proteins derived from the virus. The proteins that constitute the HCV envelope comprise envelope protein 1 (referred to as "E1"), envelope protein 2 (referred to as "E2") and p7. In particular, E1 and E2 each have a transmembrane region at the C terminus, and E1 and E2 are anchored to the HCV membrane through such transmembrane region. Thus, the E1 and the E2 proteins of HCV are exposed to the outside, and HCV adheres to and infects cells via E1 and/or E2.

According to phylogenetic analysis using nucleotide sequences of the HCV strain, HCV is classified into 6 types (i.e., genotypes 1 to 6) and such genotypes are further classified into several subtypes. In addition, the nucleotide sequences of the full-length genomes of a plurality of HCV genotypes are determined (Simmonds, P. et al., Hepatology, 10: 1321-1324, 1994; Choo, Q. L. et al., Science, 244: 359-362, 1989; Okamoto, H. et al., J. Gen. Virol., 73: 673-679, 1992; and Mori, S. et al., Biochem. Biophys. Res. Commun. 183: 334-342, 1992). Specific examples of known strains are as follows: the HCV strain of genotype 1a (e.g., the H77 strain (GenBank Accession No. AF011751)); the HCV strains of genotype 1b (e.g., the J1 strain (GenBank Accession No. D89815), the Con1 strain (GenBank Accession No. AJ238799; it may be referred to as the Con-1 or con1 strain), and the TH strain (Wakita, T. et al., J. Biol. Chem., 269, 14205-14210, 1994, JP Patent Publication (kokai) No. 2004-179 A)); and the HCV strains of genotype 2a (e.g., the JFH-1 strain (GenBank Accession No. AB047639); it may be referred to as the JFH1 strain), the J6CF strain (GenBank Accession No. AF177036), the JCH-1 strain (GenBank Accession No. AB047640), the JCH-2 strain (GenBank Accession No. AB047641), the JCH-3 strain (GenBank Accession No. AB047642), the JCH-4 strain (GenBank Accession No. AB047643), the JCH-5 strain (GenBank Accession No. AB047644), and the JCH-6 strain (GenBank Accession No. AB047645)). In addition, the HC-J8 strain (GenBank Accession. No. D01221) is known as the HCV strain of genotype 2b, the NZL1 strain (GenBank Accession No. D17763) and the S52 strain (GenBank Accession No.) are known as the HCV strains of genotype 3a, the Tr-Kj strain (GenBank Accession No. D49374) is known as the HCV strain of genotype 3b, and the ED43 strain (GenBank Accession No.) is known as the HCV strain of genotype 4a, for example. The list of the GenBank Accession numbers of other strains has already been reported (Tokita, T. et al., J. Gen. Virol., 79: 1847-1857, 1998; Cristina, J. & Colina, R., Virol. J., 3: 1-8, 2006).

The genomic nucleotide sequences of the JFH-1 strain and HCV strains other than the JFH-1 strain of the present invention are available from the above documents or GenBank, HCV strains other than the JFH-1 strain of the aforementioned genotypes may be selected, and a strain of genotype 1a, 1b, or 2a is preferable.

The chimeric HCV gene can be prepared by performing PCR using the vector prepared via cloning of cDNA of HCV genome RNA as a template and synthesized DNA as a primer, amplifying necessary regions of the HCV genes, and ligating the resultants.

Further, cDNA of the chimeric HCV gene is ligated to an adequate restriction site located downstream of a promoter (e.g., the T7 promoter) of the pJFH1 plasmid (Wakita, T. et al., Nat. Med., 11:791-796, 2005, WO 2004/104198) to prepare a vector used for synthesizing HCV genome RNA. Upon introduction of RNA transcribed from such vector into a cell such as the Huh-7 cell, virus replication and packaging take place, and infectious HCV particles can then be produced.

(2) HCV Particle Preparation

RNA may be synthesized from HCV cDNA cloned under the control of a promoter, and the resulting RNA may be introduced into a cell to prepare a chimeric HCV particle.

Specifically, chimeric HCV particles can be prepared by a method comprising a step of culturing a cell that produces such HCV particles and a step of recovering such HCV particles. The cell that produces HCV particles can be obtained by infecting an HCV-sensitive cell (i.e., a cell capable of producing HCV particles) with the chimeric HCV particles of the present invention.

Examples of the promoter include, but are not limited to, T7 promoter, SP6 promoter, and T3 promoter, with T7 promoter being preferable.

A method for preparing the RNA in vitro using, as a template, a nucleic acid into which HCV cDNA has been cloned under the control of T7 promoter can be carried out with the use of, for example, the MEGAscript T7 kit (Ambion).

Cells into which RNA is introduced may be any cells that are capable of producing HCV particles, and examples include Huh-7, HepG2, IMY-N9, HeLa, 293 and 293T cells or cells derived from any thereof. Examples of preferable cells include Huh-7 cells or Huh7.5 cells derived therefrom and Huh7.5.1 cells. In addition, cells which express CD81 and/or Claudin 1 genes in Huh-7, HepG2, IMY-N9, HeLa, 293, or 293T cells may also be used (Lindenbach, B. D. et al., Science, 309: 623-626, 2005; Evans, M. J. et al., Nature, 446: 801-805, 2007; and Akazawa, D. et al., J. Virol., 81: 5036-5045, 2007).

Examples of methods for introducing RNA into cells include calcium phosphate coprecipitation, a DEAE-dextran method, lipofection, microinjection, and electroporation. Lipofection and electroporation are preferable, and electroporation is more preferable.

When cDNA is introduced into cells, HCV cDNA may be expressed in a system involving the use of an RNA polymerase I promoter and a terminator (WO 27037428 A1).

The capacity of the cells for virus particle production can be detected with the use of antibodies to proteins that constitute an HCV particle released in culture solution, such as a Core protein, an E1 protein and an E2 protein. Also, HCV genome RNA contained in the HCV virus particles in a culture medium may be amplified via RT-PCR using specific primers to detect the HCV genome RNA of interest, so that the presence of HCV virus particles can be indirectly detected.

Whether or not the prepared viruses are infectious can be evaluated by culturing cells into which HCV RNA has been introduced, bringing the resulting supernatant into contact with HCV permissive cells (e.g., Huh-7 cells or derivative strains thereof), and immunologically staining the cells with an anti-Core antibody, for example, after 48 hours to count the number of infected cells. Alternatively, the evaluation can be carried out by subjecting a cell extract to electrophoresis on SDS-polyacrylamide gel and detecting core proteins via Western blotting.

(3) Acquisition of Particle-Producing Cell Line

For efficient replication of the HCV genome, it is necessary that a mutation occur in the nucleotide sequence of the genome (Lohmann, V. et al., J. Virol. 75: 1437-1449, 2001). A mutation that enhances replication is referred to as an "adaptive mutation." The cells into which the HCV genome RNA has been introduced as prepared in (2) above may be subjected to subculture to obtain cell lines that continuously produce HCV particles. By continuing such culture, an adaptive mutation may occasionally take place in the HCV genome, and the production of HCV particles may be significantly enhanced.

A typical example of the use of such phenomenon is a technique in which genomic RNA of the chimeric HCV is introduced into a cell and a mutant exhibiting the improved ability to produce the virus is selected. An example of such mutation is permissive mutation of chimeric HCV particles of the H77 strain and the JFH-1 strain (MinKyung Y. et al., J. Virol., 81: 629-638, 2007). Permissive mutation randomly takes place depending on the virus strain, design of the chimeric HCV genome (the construct), and conditions of experimentation. Accordingly, such mutation is not necessarily applicable to genotype 1b. This necessitates the performance of an experiment for each construct of interest to obtain permissive mutants.

A mutation of a single amino acid residue significantly changes the replication capacity of HCV and the ability to produce the HCV particle. Mutations vary depending on the HCV genotype, the type of cell used for culture, and the experiment. Since nucleic acid mutation that is necessary for a mutation of a single amino acid residue cannot be detected via hybridization, the HCV gene sequence must be subjected to sequencing in order to detect such mutation.

Thus, an HCV genome sequence that is capable of producing a large quantity of HCV particles can be found by isolating HCV genome RNA from such cells and determining the nucleotide sequence.

In order to inspect whether or not such mutation is associated with the capacity for HCV replication or ability to produce the HCV particle, further, it is necessary to introduce a mutation into the original HCV genome and inspect whether or not the capacity for HCV replication or ability to produce the HCV particle is reproduced. In order to introduce a mutation into the original HCV genome, PCR may be carried out, or a commercially available mutagenesis kit (e.g., KOD-Plus-Mutagenesis Kit, Toyobo Co., Ltd.) may be used.

In addition, whether or not such mutation is specific for the HCV genome used or is effective for other HCV genomes can also be confirmed by reintroducing a mutation into an HCV genome that has not experienced mutation.

In the present invention, when the amino acid residue at the N terminus of the Core protein of the TH/JFH-1 strain (Example 1 below) was designated as amino acid residue 1, a mutation from proline residue, the 328th amino acid residue (amino acid 328) (or the 137th amino acid (amino acid 137) when the amino acid residue at the N terminus of the E1 protein was designated as amino acid residue 1) into alanine or threonine is found. The 328th amino acid residue from the amino acid residue at the N terminus of the Core protein is the same in the Con1 strain (GenBank Accession No. AJ238799) and the J1 strain (GenBank Accession No. D89815) of genotype 1b, the H77 strain of genotype 1a (GenBank Accession No. AF011751), the JFH-1 strain of genotype 2a (GenBank Accession No. AB047639), and the J6CF strain (GenBank Accession No. AF177036), in addition to the TH strain. It is suggested that a mutation of proline into another amino acid, and preferably a mutation thereof into alanine or threonine, would be effective for any HCV strain having proline as the 328th amino acid residue from the amino acid residue at the N terminus of the Core protein, in addition to the TH strain.

(4) Use of HCV Particles

HCV particles are preferable for applications as vaccines and antigens used for preparing anti-HCV antibodies.

Specifically, HCV particles can be used as vaccines without modification, but HCV particles can be attenuated or inactivated via a method known in the art. The virus can be inactivated by adding and mixing an inactivator such as formalin, .beta.-propiolactone, or glutardialdehyde in, for example, a virus suspension and allowing the inactivator to react with the virus (Appaiahgari, M. B. & Vrati, S., Vaccine, 22: 3669-3675, 2004). Attenuated vaccines can be obtained by infecting cultured animal cells or animals (excluding humans) with chimeric HCV particles and repeating subculture to attenuate the pathogenicity. Alternatively, pathogenicity can be attenuated by negatively modifying a region associated with HCV proliferation or infection, for example, the Core-NS5B region, via genetic engineering.

Thus, the HCV vaccine of the present invention can be produced by a method comprising a step of inactivating or attenuating the chimeric HCV particles of the present invention to prepare the inactivated or attenuated chimeric HCV particles and a step of preparing the inactivated or attenuated chimeric HCV particles into the form of HCV vaccines.

The vaccine of the present invention can be formulated into a dosage form of, for example, a solution or suspension. The vaccine can be prepared in a solid state (e.g., a lyophilized preparation) that is suitable for dissolution or suspension it in a solution, so that the vaccine can be reconstituted immediately before use. Alternatively, such solid or preparation can be emulsified in the presence of a pharmaceutical surfactant or encapsulated in liposomes.

Active immunogenic ingredients, such as HCV particles, are often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredients. Examples of adequate excipients include water, physiological saline, dextrose, glycerol, ethanol, and mixtures thereof.

Further, the vaccine can contain a minor amount of an auxiliary agent (e.g., a humidifier or emulsifier), a pH buffer, and/or an adjuvant that enhances vaccine efficacy, where needed.

Examples of the effective adjuvant include, but are not limited to, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (referred to as CGP11637 or nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dip- almitoyl-sn-glycero-3-hydr oxyphosphoryloxy)-ethylamine (referred to as CGP19835A or MTP-PE), and RIBI. RIBI contains three components extracted from bacteria; i.e. monophosphoryl lipid A, trehalose dimycolate, and a cell wall skeleton (HPL+TDM+CWS), in 2% squalene/Tween.RTM. 80 emulsion.

Efficacy of an adjuvant can be determined by assaying the amount of antibodies resulting from administration of a vaccine comprising HCV particles to a mammalian animal.

The vaccine of the present invention is generally administered parenterally, by injection such as subcutaneous injection or intramuscular injection, for example. Examples of other formulations that are suitable as other forms of dosage include suppositories and, optionally, oral preparations.

Optionally, one or more compounds having adjuvant activity can be added to the HCV vaccine. An adjuvant is a non-specific stimulant to the immune system. Such substance enhances the immune response of a host against HCV vaccines. Specific examples of adjuvants that are known in the art include Freund's complete and incomplete adjuvants, vitamin E, a nonionic block copolymer, muramyl dipeptide, saponin, mineral oil, vegetable oil, and Carbopol. Examples of adjuvants that are particularly suitable for mucosal application include E. coli thermolabile toxin (LT) and Cholera toxin (CT). Examples of other adequate adjuvants include aluminum hydroxide, aluminum phosphate or aluminum oxide, an oil emulsion (e.g., Bayol.RTM. or Marcol 52.RTM.), saponin, and a vitamin E solubilizate. Accordingly, the vaccine of a preferable embodiment of the present invention comprises an adjuvant.

Concerning an injectable solution for subcutaneous, intracutaneous, intramuscular, or intravenous administration, other specific examples of a pharmaceutically acceptable carrier or diluent that is used for administration in combination with the HCV vaccine of the present invention in the injectable solution include a stabilizer, a carbohydrate (e.g., sorbitol, mannitol, starch, sucrose, glucose, or dextran), a protein (e.g., albumin or casein), a protein-containing substance (e.g., bovine serum or skimmed milk), and buffer (e.g., phosphate buffer).

Examples of conventional binders and carriers that are used for suppositories include polyalkylene glycol and triglyceride. Such suppositories can be prepared from a mixture comprising 0.5% to 50%, and preferably 1% to 20% active ingredients by weight. Oral preparations comprise excipients that are generally used. Examples of excipients include mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, and magnesium carbonate of pharmaceutical grade.

The vaccine of the present invention can be in the form of a solution, suspension, tablet, pill, capsule, sustained-release preparation, or powder, and its active ingredients (virus particles or part thereof) account for 10% to 95%, and preferably 25% to 70% thereof by weight.

The vaccine of the present invention is administered in a manner suitable for a dosage form and in an amount that can exert preventive and/or therapeutic effects. The amount to be administered is generally 0.01 .mu.g to 100,000 .mu.g antigen per dose. Such amount varies depending on the patient to be treated, the capacity of the patient for antibody synthesis in the immune system, and the desired degree of protection. Also, the amount varies depending on the route of administration, such as oral, subcutaneous, intracutaneous, intramuscular, or intravenous administration.

The vaccine of the present invention can be administered according to a single-administration schedule, and preferably according to a multiple-administration schedule. In the case of a multiple-administration schedule, 1 to 10 separate administrations are performed at the time of initiation of inoculation, and another administration can then be performed with a time interval that is necessary for maintaining and/or enhancing the immune response. For example, the second administration can be performed 1 to 4 months after the first. Where needed, administration may be subsequently performed several months after the first. The administration regimen is, at least partially, determined according to the needs of individual, and the regimen depends on the judgment made by a doctor.

Further, the vaccine comprising the HCV particles of the present invention may be administered with another immunosuppressant agent (e.g., immunoglobulin).

Further, the present invention provides a method in which the vaccine comprising the HCV particles of the present invention is administered to a healthy individual to induce an immune response to HCV in such healthy individual, and the vaccine is used for preventing new HCV infection. The present invention also provides a method in which the vaccine comprising the HCV particles of the present invention is administered to a patient infected with HCV to induce a potent immune response to HCV in vivo, and the vaccine is thus used as a therapeutic vaccine that eliminates HCV.

The HCV particles of the present invention are also useful as antigens used for preparing antibodies. The antibodies that recognize the HCV particles of the present invention used as antigens can be used for preventing or treating HCV infection as passive immunotherapeutic agents. Any antibodies can be used without limitation, and examples thereof include polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, fragments of any thereof (e.g., Fc, Fab and (Fab').sub.2), single-stranded antibodies (e.g., scFv), camel antibodies, and polyvalent antibodies (e.g., divalent and trivalent antibodies). Any antibodies can be used, and examples include IgG, IgE, IgM, IgD, IgA and IgY antibodies. Examples of the classes thereof include IgG1 to IgG4 and IgA1 to IgA2. Further, antibodies may involve chemical modification, such as glycosylation, PEG-modification, acetylation, phosphorylation, or amidation.

The anti-HCV antibodies can be prepared by a method comprising a step of administering the chimeric HCV particles, which are or are not inactivated or attenuated, of the present invention to animals (excluding humans), and preferably to mammalians or birds.

Examples of mammalians include mice, rats, rabbits, goats, sheep, horses, cattle, guinea pigs, dromedaries, Bactrian camels, and lamas. Dromedaries, Bactrian camels, and lamas are suitable for preparing an antibody consisting of the H chain. Examples of birds include chickens, geese and ostriches.

The blood serum may be taken from an animal to which the HCV particles of the present invention have been administered, in order to obtain antibodies of interest by well-known methods. Examples of such methods include ammonium sulfate fractionation, ion exchange chromatography, Protein A or Protein G-binding affinity chromatography, and gel filtration chromatography.

In addition, hybridomas that produce monoclonal antibody-producing cells can be prepared with the use of cells or tissue (e.g., B cells, spleen cells and lymph nodes) of the animals immunized with the HCV particles of the present invention and myeloma cells (e.g., myeloma cells derived from mice or rats). Methods for producing hybridomas are well-known in the art, and the method described in, for example, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, 1988) or Tan Kuron Kotai Jikken Manual (Manual of Monoclonal Antibody Experiments), Toyama and Ando (ed.), Kodansha Ltd., 1987) can be employed.

Monoclonal antibody-producing cells may be prepared via cell fusion or via other methods involving introduction of DNA of a cancer gene or infection with Epstein-Barr for immobilization of B lymphocytes.

Humanized antibodies and human antibodies can be prepared via phage display (e.g., Brinkman et al., J. Immunol. Methods, 182: 41-50, 1995; Ames et al., J. Immunol. Methods, 184: 177-186, 1995; WO 98/46645; WO 98/50433; and WO 98/24893) or with the use of human antibody-producing mice (e.g., KM mice (Kirin Pharma Co., Ltd.) or Xeno mice (Abgenix/Amgen)).

The monoclonal, polyclonal, human, or humanized antibodies obtained by such techniques are useful for diagnosis, therapy, and prevention of HCV.

The antibodies prepared with the use of the HCV particles of the present invention are administered with pharmaceutically acceptable solubilizers, additives, stabilizers, buffers, or the like. Such antibodies may be administered via any route. Subcutaneous, intracutaneous, or intramuscular administration is preferable, and intravenous administration is more preferable.

A preferable example of an antibody prepared with the use of the HCV particles of the present invention is an anti-hepatitis C virus antibody that recognizes the chimeric hepatitis C virus (HCV) particles of the present invention (i.e., the chimeric HCV particles comprising the nucleic acid of the present invention as the virus genome) as antigens. Such anti-hepatitis C virus antibodies can be prepared so as to react with the chimeric HCV particles of the present invention. Such anti-hepatitis C virus antibodies can bind to (react with) not only the chimeric HCV particles of the present invention, but they can also bind to (react with) a wide variety of other hepatitis C virus particles to inhibit the functions thereof, regardless of the process for producing the same.

In addition, the HCV particles of the present invention (i.e., the chimeric HCV particles) or cells that produce such particles can be used for screening for anti-HCV substances.

Specifically, this method for screening for an anti-hepatitis C virus substance comprises culturing, in the presence of a test substance:

(a) the cell that produces chimeric HCV particles; or

(b) the chimeric HCV particle and hepatitis C virus-sensitive cell, and detecting replicon RNA or a virus particle derived from the above-mentioned nucleic acid contained in the chimeric HCV particles of the present invention in the culture product.

According to the above method, anti-HCV substances are selected as those capable of inhibiting virus infection or proliferation. In the present invention, the term "replicon RNA" mainly refers to RNA capable of autonomous replication, which is prepared via modification of the HCV virus genome. The term "capable of autonomous replication" used herein refers to the capacity for autonomously reproducing (i.e., replicating) a nucleic acid copy in a cell, as with plasmid DNA. An example of a known subgenome replicon RNA is recombinant RNA prepared via recombination of the translational region of the HCV structural protein with a drug-resistant gene and insertion of IRES of EMCV (encephalomyocarditis virus) into a site downstream of such recombined region. RNA replication is observed in cells into which such recombinant RNA has been introduced. The term "full-length genome replicon RNA" refers to RNA capable of autonomous replication of RNA derived from the full-length HCV genome when it has been introduced into cells. A typical example is recombinant RNA prepared by inserting a drug resistant gene (or a reporter gene) and IRES into a space between the 5'-untranslated region and the gene encoding the HCV core protein of RNA derived from the full-length HCV genome. The term "replicon RNA derived from a nucleic acid" used in the above method refers to replicon RNA transcribed from such nucleic acid. Examples of hepatitis C virus-sensitive cells include, but are not limited to, cells exemplified as cells into which HCV-derived RNA is to be introduced in the section "(2) HCV particle preparation" above (e.g., Huh-7, HepG2, IMY-N9, HeLa, 293 and 293T cells or derivative cells thereof).

EXAMPLES

Hereafter, the present invention is described in greater detail with reference to the examples, although the technical scope of the present invention is not limited thereto. In the examples below, the TH strain is exemplified as an HCV strain other than the JFH-1 strain, and strains other than the designated strain can also be prepared.

Example 1

Construction of TH/JFH-1 Plasmid

As cDNA of HCV genome RNA, cDNA of the TH/JFH-1 chimera comprising 5' UTR that is from the JFH-1 strain of genotype 2a (GenBank Accession No. AB047639, Kato, T. et al., Gastroenterology, 125: 1808-1817, 2003), a region from the Core protein to the N terminal 33 amino acids of the NS2 protein that is from the TH strain of genotype 1b (Wakita, T. et al., J. Biol. Chem., 269: 14205-14210, 1994; Moradpour, D. et al., Biochem. Biophys. Res. Commun., 246: 920-924, 1998; and WO 2006/022422), and a region from the N-terminal amino acid 34 of NS2 to 3' UTR that is from the JFH-1 strain of genotype 2a, was prepared.

SEQ ID NO: 5 shows the amino acid sequence of a protein encoded by TH/JFH-1. FIG. 1 shows a method for preparing such plasmid.

Specifically, cDNA corresponding to the entire genome RNA region derived from the JFH-1 strain was cloned into the pUC19 plasmid, and the resulting plasmid DNA, pJFH1 (Wakita, T. et al., Nat. Med., 11: 791-796, 2005; WO 2004/104198), and pTH comprising part of the virus genome TH strain isolated from a hepatitis C patient (WO 2006/022422) were used.

pJFH1 was used as a template, 10 .mu.l of 5.times. buffer and 1 .mu.l of the 10 mM dNTP mixture attached to the Phusion High-Fidelity DNA Polymerase kit (FINNZYMES), and 2.5 .mu.l each of 10 .mu.M primers -21M13 (SEQ ID NO: 8) and MS98 (SEQ ID NO: 9) were added, and deionized water was added to bring the total amount to 49.5 .mu.l in the end. Thereafter, 0.5 .mu.l of Phusion DNA Polymerase (FINNZYMES) was added thereto, and PCR was carried out. PCR was carried out by repeating a cycle of 98.degree. C. for 10 seconds, 58.degree. C. for 30 seconds, and 72.degree. C. for 45 seconds 30 times. The resulting PCR product was designated as PCR product No. 1.

Subsequently, pTH was used as a template, 10 .mu.l of 5.times. buffer and 1 .mu.l of the 10 mM dNTP mixture attached to the Phusion High-Fidelity DNA Polymerase kit (FINNZYMES), and 2.5 .mu.l each of 10 .mu.M primers MS97 (SEQ ID NO: 10) and MS96 (SEQ ID NO: 11) were added, and deionized water was added to bring the total amount to 49.5 .mu.l in the end. Thereafter, 0.5 .mu.l of Phusion DNA Polymerase (FINNZYMES) was added thereto, and PCR was carried out. PCR was carried out by repeating a cycle of 98.degree. C. for 10 seconds, 58.degree. C. for 30 seconds, and 72.degree. C. for 45 seconds 30 times. The resulting PCR product was designated as PCR product No. 2.

Subsequently, pJFH1 was used as a template, 10 .mu.l of 5.times. buffer and 1 .mu.l of the 10 mM dNTP mixture attached to the Phusion High-Fidelity DNA Polymerase kit (FINNZYMES), and 2.5 .mu.l each of 10 .mu.M primers MS99 (SEQ ID NO: 12) and MS89 (SEQ ID NO: 13) were added, and deionized water was added to bring the total amount to 49.5 .mu.l in the end. Thereafter, 0.5 .mu.l of Phusion DNA Polymerase (FINNZYMES) was added thereto, and PCR was carried out. PCR was carried out by repeating a cycle of 98.degree. C. for 10 seconds, 58.degree. C. for 30 seconds, and 72.degree. C. for 45 seconds 30 times. The resulting PCR product was designated as PCR product No. 3.

PCR products were purified from agarose gel and eluted with the use of 50 .mu.l of EB buffer attached to the QIAquick Gel Extraction kit (QIAGEN). DNAs of PCR product No. 1, PCR product No. 2, and PCR product No. 3 were mixed in amounts of 1 .mu.l each, the resultant was used as a template, 10 .mu.l of 5.times. buffer and 1 .mu.l of the 10 mM dNTP mixture attached to the Phusion High-Fidelity DNA Polymerase kit (FINNZYMES), and 2.5 .mu.l each of 10 .mu.M primers -21M13 (SEQ ID NO: 8) and MS89 (SEQ ID NO: 13) were added, and deionized water was added to bring the total amount to 49.5 .mu.l in the end. Thereafter, 0.5 .mu.l of Phusion DNA Polymerase (FINNZYMES) was added thereto, and PCR was carried out. PCR was carried out by repeating a cycle of 98.degree. C. for 10 seconds, 58.degree. C. for 30 seconds, and 72.degree. C. for 2 minutes 30 times. The resulting PCR product was designated as PCR product No. 4.

pJFH1 and the purified PCR product No. 4 were digested with the restriction enzymes, EcoRI and SpeI, and the DNA fragments were separated via agarose gel electrophoresis, followed by purification. These two DNA fragments were mixed with Ligation high (New England Biolabs), and the two DNA fragments were ligated to each other. The vector was designated as pTH/JFH1. This pTH/JFH1 vector is a nucleotide sequence encoding a chimeric gene having the 5'-untranslated region derived from the JFH-1 strain; a region encoding the Core, E1, E2, and p7 proteins and the N-terminal 33 amino acid residues of NS2 protein derived from the TH strain; a region encoding the NS2 protein starting at amino acid residue 34 at the N-terminus and the NS3, NS4A, NS4B, NS5A and NS5B proteins and the 3'-untranslated region of the JFH-1 strain.

Example 2

In Vitro RNA Synthesis and Introduction Thereof Into Cell

pTH/JFH1 was cleaved with XbaI, and the resultant was then subjected to phenol/chloroform extraction and ethanol precipitation. Subsequently, the XbaI cleavage fragment was treated with Mung Bean Nuclease, and an 3' terminal extra nucleotide sequence derived from the XbaI recognition sequence was removed. Further, proteinase K treatment, phenol/chloroform extraction, and ethanol precipitation were carried out to purify the DNA fragments. The cleaved plasmids were used as templates, and the reaction was allowed to proceed at 37.degree. C. for 3 hours using the MEGAscript T7 kit (Ambion) to synthesize HCV RNA. After the reaction, the synthesized RNA was treated with DNaseI and extracted with acidic phenol, followed by purification via ethanol precipitation.

The Huh7 cells (3.times.10.sup.6 cells) and 10 .mu.g of HCV RNA were suspended in 400 .mu.l of the Cytomix solution (120 mM KCl, 0.15 mM CaCl.sub.2, 10 mM K.sub.2HPO.sub.4/KH.sub.2PO.sub.4, 25 mM Hepes, 2 mM EGTA, 5 mM MgCl.sub.2, 20 mM ATP, and 50 mM Glutathione), the suspension was transferred to a 4-mm cuvette, and electroporation was carried out using the Gene Pulser (BioRad) at 260 V and 950 .mu.F. Thereafter, the cells into which HCV RNA had been introduced were seeded on a 10 cm.sup.2 dish and then subcultured.

Example 3

Production of HCV Particles with Cells into which Th/Jfh-1 RNA Had been Introduced

At the time of subculture of cells into which TH/JFH-1 RNA had been introduced, the HCV Core protein contained in the culture supernatants was quantified using the HCV antigen ELISA test kit (Ortho) to confirm the production of HCV particles. As a result, the amount of HCV Core protein in the culture supernatant continued to decrease until 23 days after the introduction with the elapse of time, such amount began to increase 26 days after the introduction, and a constantly high amount of production was observed 34 days after the introduction (FIG. 2). Thus, it was considered that TH/JFH-1 RNA did not have the high capacity for virus production when it was introduced into Huh7 cell but it would acquire a high capacity for virus production upon introduction of an adaptive mutation necessary for virus production into the virus genome.

Example 4

Analysis of HCV Genome Sequence in Cell Infected with TH/JFH-1 Virus, which Had Undergone Subculture

In order to inspect an adaptive mutation that is necessary for the THIJFH-1 virus to be produced in high amounts, total RNA was extracted from infected cells 34 days after RNA introduction, and the sequence of the HCV genome contained therein was analyzed.

Total RNA was extracted using Trizol (Invitrogen) and transcribed into cDNA. This cDNA was divided into 5 DNA fragments via PCR, ligated to the pGEM-T Easy vector (Promega), and transformed into E. coli DH5a to obtain colonies. Plasmids were extracted from 10 colonies using the QIAprep Mini kit (QIAGEN), and the nucleotide sequences of the DNA fragments were confirmed.

As a result, proline CCU (P) in the E1 region of the TH strain was found to have been substituted with alanine ACU (A) or threonine GCU (T). Proline corresponds to amino acid 328 when methionine; i.e., the amino acid residue at the N terminus of the Core protein of the TH strain (Wakita, T. et al., J. Biol. Chem., 269, 1994, pp. 14205-14210; Moradpour et al., Biochem. Biophys. Res. Commun., 246, 1998, pp. 920-924; and WO 2006/022422) was designated as amino acid residue 1 or amino acid residue 137 when the amino acid residue at the N terminus of the E1 protein was designated as amino acid residue 1.

SEQ ID NO: 6 shows the amino acid sequence of TH/JFH-1 (PA) and SEQ ID NO: 7 shows the amino acid sequence of TH/JFH-1 (PT).

Example 5

Construction of TH/JFH-1 Mutant Plasmid

A plasmid having an adaptive mutation necessary for the TH/JFH-1 virus to be produced in high amounts described in Example 4 was constructed. FIG. 3 shows a method for preparing a plasmid.

Specifically, pTH/JFH1 was used as a template, 10 .mu.l of 5.times. buffer and 1 .mu.l of the 10 mM dNTP mixture attached to the Phusion High-Fidelity DNA Polymerase kit (FINNZYMES), and 2.5 .mu.l each of 10 .mu.M primers MS151 (SEQ ID NO: 14) and MS165 (SEQ ID NO: 15) were added, and deionized water was added to bring the total amount to 49.5 .mu.l in the end. Thereafter, 0.5 .mu.l of Phusion DNA Polymerase (FINNZYMES) was added thereto, and PCR was carried out. PCR was carried out by repeating a cycle of 98.degree. C. for 10 seconds, 58.degree. C. for 30 seconds, and 72.degree. C. for 1 minute 30 times. The resulting PCR product was designated as PCR product No. 5.

Subsequently, pTH/JFH1 was used as a template, 10 .mu.l of 5.times. buffer and 1 .mu.l of the 10 mM dNTP mixture attached to the Phusion High-Fidelity DNA Polymerase kit (FINNZYMES), and 2.5 .mu.l each of 10 .mu.M primers MS164 (SEQ ID NO: 16) and MS156 (SEQ ID NO: 17) were added, and deionized water was added to bring the total amount to 49.5 .mu.l in the end. Thereafter, 0.5 .mu.l of Phusion DNA Polymerase (FINNZYMES) was added thereto, and PCR was carried out. PCR was carried out by repeating a cycle of 98.degree. C. for 10 seconds, 58.degree. C. for 30 seconds, and 72.degree. C. for 1 minute 30 times. The resulting PCR product was designated as PCR product No. 6.

PCR products were purified from agarose gel and eluted with the use of 50 .mu.l of EB buffer attached to the QIAquick Gel Extraction kit (QIAGEN). DNAs of PCR product No. 5 and PCR product No. 6 were mixed in amounts of 1 .mu.l each, the resultant was used as a template, 10 .mu.l of 5.times. buffer and 1 .mu.l of the 10 mM dNTP mixture attached to the Phusion High-Fidelity DNA Polymerase kit (FINNZYMES), and 2.5 .mu.l each of 10 .mu.M primers MS151 (SEQ ID NO: 14) and MS156 (SEQ ID NO: 17) were added, and deionized water was added to bring the total amount to 49.5 .mu.l in the end. Thereafter, 0.5 .mu.l of Phusion DNA Polymerase (FINNZYMES) was added thereto, and PCR was carried out. PCR was carried out by repeating a cycle of 98.degree. C. for 10 seconds, 58.degree. C. for 30 seconds, and 72.degree. C. for 1 minute and 30 seconds 30 times. The resulting PCR product was designated as PCR product No. 7 (FIG. 3A).

Subsequently, pTH/JFH 1 was used as a template, 10 .mu.l of 5.times. buffer and 1 .mu.l of the 10 mM dNTP mixture attached to the Phusion High-Fidelity DNA Polymerase kit (FINNZYMES), and 2.5 .mu.l each of 10 .mu.M primers MS151 (SEQ ID NO: 14) and MS163 (SEQ ID NO: 18) were added, and deionized water was added to bring the total amount to 49.5 .mu.l in the end. Thereafter, 0.5 .mu.l of Phusion DNA Polymerase (FINNZYMES) was added thereto, and PCR was carried out. PCR was carried out by repeating a cycle of 98.degree. C. for 10 seconds, 58.degree. C. for 30 seconds, and 72.degree. C. for 1 minute 30 times. The resulting PCR product was designated as PCR product No. 8.

Subsequently, pTH/JFH1 was used as a template, 10 .mu.l of 5.times. buffer and 1 .mu.l of the 10 mM dNTP mixture attached to the Phusion High-Fidelity DNA Polymerase kit (FINNZYMES), and 2.5 .mu.l each of 10 .mu.M primers MS162 (SEQ ID NO: 19) and MS156 (SEQ ID NO: 17) were added, and deionized water was added to bring the total amount to 49.5 .mu.l in the end. Thereafter, 0.5 .mu.l of Phusion DNA Polymerase (FINNZYMES) was added thereto, and PCR was carried out. PCR was carried out by repeating a cycle of 98.degree. C. for 10 seconds, 58.degree. C. for 30 seconds, and 72.degree. C. for 1 minute 30 times. The resulting PCR product was designated as PCR product No. 9.

PCR products were purified from agarose gel and eluted with the use of 50 .mu.l of EB buffer attached to the QIAquick Gel Extraction kit (QIAGEN). DNAs of PCR product No. 8 and PCR product No. 9 were mixed in amounts of 1 .mu.l each, the resultant was used as a template, 10 .mu.l of 5.times. buffer and 1 .mu.l of the 10 mM dNTP mixture attached to the Phusion High-Fidelity DNA Polymerase kit (FINNZYMES), and 2.5 .mu.l each of 10 .mu.M primers MS151 (SEQ ID NO: 14) and MS156 (SEQ ID NO: 17) were added, and deionized water was added to bring the total amount to 49.5 .mu.l in the end. Thereafter, 0.5 .mu.l of Phusion DNA Polymerase (FINNZYMES) was added thereto, and PCR was carried out. PCR was carried out by repeating a cycle of 98.degree. C. for 10 seconds, 58.degree. C. for 30 seconds, and 72.degree. C. for 1 minute and 30 seconds 30 times. The resulting PCR product was designated as PCR product No. 10 (FIG. 3B).

pTH/JFH1 and the purified PCR product No. 7 were digested with the Acc65I restriction enzyme and the DNA fragments were separated via agarose gel electrophoresis, followed by purification. These two DNA fragments were mixed with Ligation high (New England Biolabs), and the two DNA fragments were ligated to each other. The vector was designated as pTH/JFH-1(PA). This pTH/JFH1 (PA) vector comprises a nucleotide sequence encoding a chimeric gene having the 5'-untranslated region derived from the JFH-1 strain; a region encoding the Core, E1, E2 and p7 proteins and the N-terminal 33 amino acid residues of NS2 protein derived from the TH strain; a region encoding the NS2 protein starting at amino acid residue 34 at the N-terminus and the NS3, NS4A, NS4B, NS5A and NS5B proteins, and the 3'-untranslated region of the JFH-1 strain, wherein amino acid 328, when methionine as the amino acid at the N terminus of the Core protein is designated as amino acid 1, is alanine.

Subsequently, pTH/JFH1 and the purified PCR product No. 10 were digested with the Acc65I restriction enzyme and the DNA fragments were separated via agarose gel electrophoresis, followed by purification. These two DNA fragments were mixed with Ligation high (New England Biolabs), and the two DNA fragments were ligated to each other. The vector was designated as pTH/JFH1(PT). This pTH/JFH1 (PT) vector comprises a nucleotide sequence encoding a chimeric gene having the 5'-untranslated region derived from the JFH-1 strain; a region encoding the Core, E1, E2, and p7 proteins and the N-terminal 33 amino acid residues of NS2 protein derived from the TH strain; a region encoding the NS2 protein starting at amino acid residue 34 at the N-terminus and the NS3, NS4A, NS4B, NS5A and NS5B proteins, and the 3'-untranslated region of the JFH-1 strain, wherein amino acid 328, when methionine as the amino acid at the N terminus of the Core protein is designated as amino acid 1, is threonine.

The nucleotide sequences of pTH/JFH1 (PA) and pTH/JFH1 (PT) are shown in SEQ ID NOs: 1 and 2, respectively, in the sequence listings.

Example 6

Preparation of TH/JFH-1 (PA) and TH/JFH-1 (PT) Viruses

Plasmids prepared in Example 5 were cleaved with XbaI, and the resultants were then subjected to phenol/chloroform extraction and ethanol precipitation. Subsequently, the XbaI cleavage fragments were treated with Mung Bean Nuclease, and the 3'-terminal extra nucleotide sequences derived from the XbaI recognition sequence were removed. Further, proteinase K treatment, phenol/chloroform extraction, and ethanol precipitation were carried out to purify the DNA fragments. The cleaved plasmids were used as templates to synthesize HCV RNAs using the MEGAscript T7 kit (Ambion).

The Huh-7 cells (3.times.10.sup.6 cells) and 10 .mu.g of HCV RNA were suspended in 400 .mu.l of the Cytomix solution (120 mM KCl, 0.15 mM CaCl.sub.2, 10 mM K.sub.2HPO.sub.4/KH.sub.2PO.sub.4, 25 mM Hepes, 2 mM EGTA, 5 mM MgCl.sub.2, 20 mM ATP, and 50 mM Glutathione), the suspension was transferred to a 4-mm cuvette, and electroporation was carried out using the Gene Pulser (BioRad) at 260 V and 950 .mu.F. Thereafter, the cells into which HCV RNA had been introduced were seeded on a 10 cm.sup.2 dish and then subcultured.

At the time of subculture of cells into which TH/JFH-1 RNA, TH/JFH-1 (PA) RNA (SEQ ID NO: 3), and TH/JFH-1 (PT) RNA (SEQ ID NO: 4) prepared in Example 2 had been introduced, the HCV Core protein contained in the culture supernatants were quantified using the HCV antigen ELISA test kit (Ortho) to confirm the production of HCV particles. From the initial stage to the late stage of culture, the amount of HCV Core protein contained in the culture supernatant of cells into which RNA without mutation had been introduced was compared with that into which RNA with mutation had been introduced. As a result, the latter amount was found to be higher (FIG. 4).

Example 7

Evaluation of Infectivity of Virus into which Mutation Had been Introduced

The infectivity of viruses produced from cells into which TH/JFH-1(PA) RNA had been introduced was compared with that of wild-type TH/JFH-1. Changes in the amount of HCV Core protein in the cells and in the culture supernatants 4, 24, 48, 72 and 96 hours after RNA introduction were analyzed, and the infectivity of the culture supernatant was analyzed.

Specifically, TH/JFH-1 and TH/JFH-1(PA) RNA were synthesized in the same manner as in Example 6, 3.times.10.sup.6 Huh-7 cells and 10 .mu.g of HCV RNA were suspended in 400 .mu.l of the Cytomix solution (120 mM KCl, 0.15 mM CaCl.sub.2, 10 mM K.sub.2HPO.sub.4/KH.sub.2PO.sub.4, 25 mM Hepes, 2 mM EGTA, 5 mM MgCl.sub.2, 20 mM ATP, and 50 mM glutathione), the suspension was transferred to a 4-mm cuvette, and electroporation was carried out using the Gene Pulser (BioRad) at 260 V and 950 .mu.F. Thereafter, the cells into which HCV RNA had been introduced were seeded on a 10 cm.sup.2 dish, the culture supernatant was collected 4, 24, 48, 72, and 96 hours later, the collected culture supernatant was filtered through a 0.45-.mu.m filter (Millipore), and the HCV Core proteins were quantified using the HCV antigen ELISA test kit (Ortho). The 10 cm.sup.2 dish from which the culture supernatant had been removed was washed with PBS, cells were scraped off using 500 .mu.l of PBS and a scraper (Sumitomo Bakelite Co., Ltd.), and the cells were recovered via centrifugation. Passive Lysis Buffer (100 .mu.l Promega) was added to the recovered cells to prepare a lysate, and the HCV Core proteins contained therein were quantified using the HCV antigen ELISA test kit (Ortho) as in the case of the culture supernatant.

The culture supernatant was serially diluted with a culture medium to quantify the infectious titer thereof in the following manner. The Huh-7 cells were seeded on a 96-well polylysine-coated plate (Corning) at 1.times.10.sup.4 cells/well, culture was conducted for a whole day, the culture medium was exchanged with the culture supernatant which had been serially diluted with a culture medium, and culture was conducted for an additional 3 days. Thereafter, the culture medium was discarded, the cells were washed 3 times with PBS, and the cells were immobilized with methanol for 15 minutes. Subsequently, the wells were blocked with the use of Block Ace (Dainippon Sumitomo Pharma Co., Ltd.) containing 0.3% Triton-X 100 and allowed to react with the anti-HCV Core-specific antibodies (clone 2H9). Subsequently, the wells were washed with PBS and the cells were allowed to react with the Alexa488-labeled anti-mouse IgG antibodies (Invitrogen). Thereafter, the wells were washed with PBS, the number of infectious foci of each well was counted under a fluorescent microscope (Olympus Corporation), and the infectious titer of each culture supernatant was calculated in terms of the focus-forming unit (FFU).

As a result, the rate of the HCV Core protein secretion into the culture supernatant and the infectious titer of the culture supernatant were found to be higher in the cells into which TH/JFH-1(PA) RNA had been introduced than in cells into which wild-type TH/JFH-1 had been introduced (FIGS. 5 and 6). It was demonstrated that a mutation of proline 328 in TH/JFH-1 (SEQ ID NO: 5) would enhance HCV particle production.

INDUSTRIAL APPLICABILITY

HCV particles that are provided by the method of the present invention exhibit high levels of expression, ability to be produced, and infectivity. Thus, such HCV particles can be preferably used for preventive or therapeutic vaccines for HCV. In addition, the HCV particles of the present invention can be used as tools for inducing antibodies reacting with HCV.

All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

SEQUENCE LISTINGS

1

1919669DNAArtificialpTH/JFH1(PA) 1acctgcccct aataggggcg acactccgcc atgaatcact cccctgtgag gaactactgt 60cttcacgcag aaagcgccta gccatggcgt tagtatgagt gtcgtacagc ctccaggccc 120ccccctcccg ggagagccat agtggtctgc ggaaccggtg agtacaccgg aattgccggg 180aagactgggt cctttcttgg ataaacccac tctatgcccg gccatttggg cgtgcccccg 240caagactgct agccgagtag cgttgggttg cgaaaggcct tgtggtactg cctgataggg 300cgcttgcgag tgccccggga ggtctcgtag accgtgcacc atgagcacga atcctaaacc 360tcaaagaaaa accaaacgta acaccaaccg ccgcccacag gacgtcaagt tcccgggcgg 420tggccagatc gttggtggag tttacctgtt gccgcgcagg ggccccaggt tgggtgtgcg 480cgcgactagg aagacttccg agcggtcgca acctcgtgga aggcgacaac ctatccccaa 540ggatcgccga cccgagggca gggcctgggc tcagcctggg tacccttggc ccctctatgg 600caacgagggc atggggtggg caggatggct cctgtcaccc cgtggctccc ggcctagttg 660gggccccaat gacccccggc gcaggtcgcg taatttgggt aaagtcatcg atacccttac 720atgcggcttc gccgacctca tggggtacat tccgctcgtc ggcgctccct tggggggcgc 780tgccagggcc ttggcgcatg gcgtccgggt tctggaggac ggcgtgaact atgcaacagg 840gaatctgccc ggttgctctt tctctatctt cctcttggct ctgctgtcct gtctaaccat 900cccagcttcc gcttatgaag tgcgcaacgt gtccggggtg taccatgtca cgaacgactg 960ctccaactcg agcattgtgt acgagacagg ggacatgatt atgcacaccc ctgggtgcgt 1020gccctgtgtt cgggagaaca actcctcccg ctgctgggca gcgctcactc ccacgctcgc 1080ggccaggaac gccagcgtcc ccaccacgac aatacggcgc cacgtcgatt tgctcgttgg 1140ggcggctgct ttctgctccg ctatgtacgt gggggatctc tgcggatctg ttttcctcgt 1200ctcccagttg ttcaccttct cgcctcgccg gcatgagaca gtgcaggact gcaattgttc 1260aatctatccc ggccacgtat caggtcaccg catggcttgg gatatgatga tgaactggtc 1320agctacaaca gccctactgg tatcgcagtt actccggatc ccacaagccg tcgtggacat 1380ggtggcgggg gcccactggg gagtcctggc gggccttgcc tactattcca tggcggggaa 1440ctgggctaag gttttgattg tgctgctact ctttgccggc gttgatgggg cgacctacgt 1500gacggggggg tcggaagcca gaggggcctc tggcttagca aacctctttt catttggggc 1560gtctcagaag atccagctca taaataccaa cggcagttgg cacatcaata gaactgccct 1620gaactgcaat gactccctcc acactgggtt tcttgccgcg ctattctaca cacacaaatt 1680caacgcgtcc ggatgtccag agcgcatggc cagctgccgc cccattgaag agttcgctca 1740ggggtatggt cccatcactt atgctgagcc ctccccctcg gaccagaggc cctattgctg 1800gcactacgcg cctcgaccgt gtggtatcat acccgcgtcg caggtgtgtg gtccagtgta 1860ctgcttcacc ccaagccctg ttgtggtggg gacgaccgat cgctccggtg cccccacgta 1920taattggggg gcgaatgaga cggacgtgct gtatctcaac aacacgcggc cgccgcaagg 1980caactggttc ggctgcacat ggatgaatgg caccgggttc accaagacgt gcgggggccc 2040cccgtgcaac atcggggggg gcggcaacaa caacaccttg acctgcccca cggactgttt 2100ccggaaacac cccgaggcca cctacaccaa atgtggttcg ggaccttggt tgacacctag 2160gtgcatggtc gactacccat acaggctctg gcactacccc tgcaccgtta actttaccat 2220ctttaaggtt aggatgtacg tgggaggtgt ggagcacagg ctcaacgccg catgcaattg 2280gacccgagga gagcgttgta acttagagga cagggataga tcagagctta gcccgctgct 2340gctgtcaaca acagagtggc aggtgctacc ttgttccttc accaccctac cggctctgtc 2400cactggtttg atccatctcc accagaacat cgtggacgtg caatacctgt acggtatagg 2460gtcggcggtt gtctcctatg caatcaaatg ggaatatgtc ttgttgctct tcctcctcct 2520ggcagacgcg cgcgtctgcg cctgcttgtg gatgatgctg ctgatagctc aagctgaggc 2580cgccttagag aacctggtgg tcctcaatgc ggcgtccctg gctggagcgc atggccttct 2640ctctttcctt gtgttcttct gtgccgcttg gtacatcaag ggcaggttga tccccggggc 2700ggcgtatgct ttttacggcg tatggccgct gctcctactc ctgctggcgt taccaccacg 2760agcatacgcc atggaccggg agatggctgc atcgtgcgga ggcgcggttt ttgtaggtct 2820ggcattcctg accttgtcac cacactataa ggcattcctc gccaagctcc tgtggtggtt 2880gtgctatctc ctgaccctgg gggaagccat gattcaggag tgggtaccac ccatgcaggt 2940gcgcggcggc cgcgatggca tcgcgtgggc cgtcactata ttctgcccgg gtgtggtgtt 3000tgacattacc aaatggcttt tggcgttgct tgggcctgct tacctcttaa gggccgcttt 3060gacacatgtg ccgtacttcg tcagagctca cgctctgata agggtatgcg ctttggtgaa 3120gcagctcgcg gggggtaggt atgttcaggt ggcgctattg gcccttggca ggtggactgg 3180cacctacatc tatgaccacc tcacacctat gtcggactgg gccgctagcg gcctgcgcga 3240cttagcggtc gccgtggaac ccatcatctt cagtccgatg gagaagaagg tcatcgtctg 3300gggagcggag acggctgcat gtggggacat tctacatgga cttcccgtgt ccgcccgact 3360cggccaggag atcctcctcg gcccagctga tggctacacc tccaaggggt ggaagctcct 3420tgctcccatc actgcttatg cccagcaaac acgaggcctc ctgggcgcca tagtggtgag 3480tatgacgggg cgtgacagga cagaacaggc cggggaagtc caaatcctgt ccacagtctc 3540tcagtccttc ctcggaacaa ccatctcggg ggttttgtgg actgtttacc acggagctgg 3600caacaagact ctagccggct tacggggtcc ggtcacgcag atgtactcga gtgctgaggg 3660ggacttggta ggctggccca gcccccctgg gaccaagtct ttggagccgt gcaagtgtgg 3720agccgtcgac ctatatctgg tcacgcggaa cgctgatgtc atcccggctc ggagacgcgg 3780ggacaagcgg ggagcattgc tctccccgag acccatttcg accttgaagg ggtcctcggg 3840ggggccggtg ctctgcccta ggggccacgt cgttgggctc ttccgagcag ctgtgtgctc 3900tcggggcgtg gccaaatcca tcgatttcat ccccgttgag acactcgacg ttgttacaag 3960gtctcccact ttcagtgaca acagcacgcc accggctgtg ccccagacct atcaggtcgg 4020gtacttgcat gctccaactg gcagtggaaa gagcaccaag gtccctgtcg cgtatgccgc 4080ccaggggtac aaagtactag tgcttaaccc ctcggtagct gccaccctgg ggtttggggc 4140gtacctatcc aaggcacatg gcatcaatcc caacattagg actggagtca ggaccgtgat 4200gaccggggag gccatcacgt actccacata tggcaaattt ctcgccgatg ggggctgcgc 4260tagcggcgcc tatgacatca tcatatgcga tgaatgccac gctgtggatg ctacctccat 4320tctcggcatc ggaacggtcc ttgatcaagc agagacagcc ggggtcagac taactgtgct 4380ggctacggcc acaccccccg ggtcagtgac aaccccccat cccgatatag aagaggtagg 4440cctcgggcgg gagggtgaga tccccttcta tgggagggcg attcccctat cctgcatcaa 4500gggagggaga cacctgattt tctgccactc aaagaaaaag tgtgacgagc tcgcggcggc 4560ccttcggggc atgggcttga atgccgtggc atactataga gggttggacg tctccataat 4620accagctcag ggagatgtgg tggtcgtcgc caccgacgcc ctcatgacgg ggtacactgg 4680agactttgac tccgtgatcg actgcaatgt agcggtcacc caagctgtcg acttcagcct 4740ggaccccacc ttcactataa ccacacagac tgtcccacaa gacgctgtct cacgcagtca 4800gcgccgcggg cgcacaggta gaggaagaca gggcacttat aggtatgttt ccactggtga 4860acgagcctca ggaatgtttg acagtgtagt gctttgtgag tgctacgacg caggggctgc 4920gtggtacgat ctcacaccag cggagaccac cgtcaggctt agagcgtatt tcaacacgcc 4980cggcctaccc gtgtgtcaag accatcttga attttgggag gcagttttca ccggcctcac 5040acacatagac gcccacttcc tctcccaaac aaagcaagcg ggggagaact tcgcgtacct 5100agtagcctac caagctacgg tgtgcgccag agccaaggcc cctcccccgt cctgggacgc 5160catgtggaag tgcctggccc gactcaagcc tacgcttgcg ggccccacac ctctcctgta 5220ccgtttgggc cctattacca atgaggtcac cctcacacac cctgggacga agtacatcgc 5280cacatgcatg caagctgacc ttgaggtcat gaccagcacg tgggtcctag ctggaggagt 5340cctggcagcc gtcgccgcat attgcctggc gactggatgc gtttccatca tcggccgctt 5400gcacgtcaac cagcgagtcg tcgttgcgcc ggataaggag gtcctgtatg aggcttttga 5460tgagatggag gaatgcgcct ctagggcggc tctcatcgaa gaggggcagc ggatagccga 5520gatgttgaag tccaagatcc aaggcttgct gcagcaggcc tctaagcagg cccaggacat 5580acaacccgct atgcaggctt catggcccaa agtggaacaa ttttgggcca gacacatgtg 5640gaacttcatt agcggcatcc aatacctcgc aggattgtca acactgccag ggaaccccgc 5700ggtggcttcc atgatggcat tcagtgccgc cctcaccagt ccgttgtcga ccagtaccac 5760catccttctc aacatcatgg gaggctggtt agcgtcccag atcgcaccac ccgcgggggc 5820caccggcttt gtcgtcagtg gcctggtggg ggctgccgtg ggcagcatag gcctgggtaa 5880ggtgctggtg gacatcctgg caggatatgg tgcgggcatt tcgggggccc tcgtcgcatt 5940caagatcatg tctggcgaga agccctctat ggaagatgtc atcaatctac tgcctgggat 6000cctgtctccg ggagccctgg tggtgggggt catctgcgcg gccattctgc gccgccacgt 6060gggaccgggg gagggcgcgg tccaatggat gaacaggctt attgcctttg cttccagagg 6120aaaccacgtc gcccctactc actacgtgac ggagtcggat gcgtcgcagc gtgtgaccca 6180actacttggc tctcttacta taaccagcct actcagaaga ctccacaatt ggataactga 6240ggactgcccc atcccatgct ccggatcctg gctccgcgac gtgtgggact gggtttgcac 6300catcttgaca gacttcaaaa attggctgac ctctaaattg ttccccaagc tgcccggcct 6360ccccttcatc tcttgtcaaa aggggtacaa gggtgtgtgg gccggcactg gcatcatgac 6420cacgcgctgc ccttgcggcg ccaacatctc tggcaatgtc cgcctgggct ctatgaggat 6480cacagggcct aaaacctgca tgaacacctg gcaggggacc tttcctatca attgctacac 6540ggagggccag tgcgcgccga aaccccccac gaactacaag accgccatct ggagggtggc 6600ggcctcggag tacgcggagg tgacgcagca tgggtcgtac tcctatgtaa caggactgac 6660cactgacaat ctgaaaattc cttgccaact accttctcca gagtttttct cctgggtgga 6720cggtgtgcag atccataggt ttgcacccac accaaagccg tttttccggg atgaggtctc 6780gttctgcgtt gggcttaatt cctatgctgt cgggtcccag cttccctgtg aacctgagcc 6840cgacgcagac gtattgaggt ccatgctaac agatccgccc cacatcacgg cggagactgc 6900ggcgcggcgc ttggcacggg gatcacctcc atctgaggcg agctcctcag tgagccagct 6960atcagcaccg tcgctgcggg ccacctgcac cacccacagc aacacctatg acgtggacat 7020ggtcgatgcc aacctgctca tggagggcgg tgtggctcag acagagcctg agtccagggt 7080gcccgttctg gactttctcg agccaatggc cgaggaagag agcgaccttg agccctcaat 7140accatcggag tgcatgctcc ccaggagcgg gtttccacgg gccttaccgg cttgggcacg 7200gcctgactac aacccgccgc tcgtggaatc gtggaggagg ccagattacc aaccgcccac 7260cgttgctggt tgtgctctcc ccccccccaa gaaggccccg acgcctcccc caaggagacg 7320ccggacagtg ggtctgagcg agagcaccat atcagaagcc ctccagcaac tggccatcaa 7380gacctttggc cagcccccct cgagcggtga tgcaggctcg tccacggggg cgggcgccgc 7440cgaatccggc ggtccgacgt cccctggtga gccggccccc tcagagacag gttccgcctc 7500ctctatgccc cccctcgagg gggagcctgg agatccggac ctggagtctg atcaggtaga 7560gcttcaacct cccccccagg gggggggggt agctcccggt tcgggctcgg ggtcttggtc 7620tacttgctcc gaggaggacg ataccaccgt gtgctgctcc atgtcatact cctggaccgg 7680ggctctaata actccctgta gccccgaaga ggaaaagttg ccaatcaacc ctttgagtaa 7740ctcgctgttg cgataccata acaaggtgta ctgtacaaca tcaaagagcg cctcacagag 7800ggctaaaaag gtaacttttg acaggacgca agtgctcgac gcccattatg actcagtctt 7860aaaggacatc aagctagcgg cttccaaggt cagcgcaagg ctcctcacct tggaggaggc 7920gtgccagttg actccacccc attctgcaag atccaagtat ggattcgggg ccaaggaggt 7980ccgcagcttg tccgggaggg ccgttaacca catcaagtcc gtgtggaagg acctcctgga 8040agacccacaa acaccaattc ccacaaccat catggccaaa aatgaggtgt tctgcgtgga 8100ccccgccaag gggggtaaga aaccagctcg cctcatcgtt taccctgacc tcggcgtccg 8160ggtctgcgag aaaatggccc tctatgacat tacacaaaag cttcctcagg cggtaatggg 8220agcttcctat ggcttccagt actcccctgc ccaacgggtg gagtatctct tgaaagcatg 8280ggcggaaaag aaggacccca tgggtttttc gtatgatacc cgatgcttcg actcaaccgt 8340cactgagaga gacatcagga ccgaggagtc catataccag gcctgctccc tgcccgagga 8400ggcccgcact gccatacact cgctgactga gagactttac gtaggagggc ccatgttcaa 8460cagcaagggt caaacctgcg gttacagacg ttgccgcgcc agcggggtgc taaccactag 8520catgggtaac accatcacat gctatgtgaa agccctagcg gcctgcaagg ctgcggggat 8580agttgcgccc acaatgctgg tatgcggcga tgacctagta gtcatctcag aaagccaggg 8640gactgaggag gacgagcgga acctgagagc cttcacggag gccatgacca ggtactctgc 8700ccctcctggt gatcccccca gaccggaata tgacctggag ctaataacat cctgttcctc 8760aaatgtgtct gtggcgttgg gcccgcgggg ccgccgcaga tactacctga ccagagaccc 8820aaccactcca ctcgcccggg ctgcctggga aacagttaga cactccccta tcaattcatg 8880gctgggaaac atcatccagt atgctccaac catatgggtt cgcatggtcc taatgacaca 8940cttcttctcc attctcatgg tccaagacac cctggaccag aacctcaact ttgagatgta 9000tggatcagta tactccgtga atcctttgga ccttccagcc ataattgaga ggttacacgg 9060gcttgacgcc ttttctatgc acacatactc tcaccacgaa ctgacgcggg tggcttcagc 9120cctcagaaaa cttggggcgc cacccctcag ggtgtggaag agtcgggctc gcgcagtcag 9180ggcgtccctc atctcccgtg gagggaaagc ggccgtttgc ggccgatatc tcttcaattg 9240ggcggtgaag accaagctca aactcactcc attgccggag gcgcgcctac tggacttatc 9300cagttggttc accgtcggcg ccggcggggg cgacattttt cacagcgtgt cgcgcgcccg 9360accccgctca ttactcttcg gcctactcct acttttcgta ggggtaggcc tcttcctact 9420ccccgctcgg tagagcggca cacactaggt acactccata gctaactgtt cctttttttt 9480tttttttttt tttttttttt tttttttttt tttttctttt tttttttttt ccctctttct 9540tcccttctca tcttattcta ctttctttct tggtggctcc atcttagccc tagtcacggc 9600tagctgtgaa aggtccgtga gccgcatgac tgcagagagt gccgtaactg gtctctctgc 9660agatcatgt 966929669DNAArtificialpTH/JFH1(PT) 2acctgcccct aataggggcg acactccgcc atgaatcact cccctgtgag gaactactgt 60cttcacgcag aaagcgccta gccatggcgt tagtatgagt gtcgtacagc ctccaggccc 120ccccctcccg ggagagccat agtggtctgc ggaaccggtg agtacaccgg aattgccggg 180aagactgggt cctttcttgg ataaacccac tctatgcccg gccatttggg cgtgcccccg 240caagactgct agccgagtag cgttgggttg cgaaaggcct tgtggtactg cctgataggg 300cgcttgcgag tgccccggga ggtctcgtag accgtgcacc atgagcacga atcctaaacc 360tcaaagaaaa accaaacgta acaccaaccg ccgcccacag gacgtcaagt tcccgggcgg 420tggccagatc gttggtggag tttacctgtt gccgcgcagg ggccccaggt tgggtgtgcg 480cgcgactagg aagacttccg agcggtcgca acctcgtgga aggcgacaac ctatccccaa 540ggatcgccga cccgagggca gggcctgggc tcagcctggg tacccttggc ccctctatgg 600caacgagggc atggggtggg caggatggct cctgtcaccc cgtggctccc ggcctagttg 660gggccccaat gacccccggc gcaggtcgcg taatttgggt aaagtcatcg atacccttac 720atgcggcttc gccgacctca tggggtacat tccgctcgtc ggcgctccct tggggggcgc 780tgccagggcc ttggcgcatg gcgtccgggt tctggaggac ggcgtgaact atgcaacagg 840gaatctgccc ggttgctctt tctctatctt cctcttggct ctgctgtcct gtctaaccat 900cccagcttcc gcttatgaag tgcgcaacgt gtccggggtg taccatgtca cgaacgactg 960ctccaactcg agcattgtgt acgagacagg ggacatgatt atgcacaccc ctgggtgcgt 1020gccctgtgtt cgggagaaca actcctcccg ctgctgggca gcgctcactc ccacgctcgc 1080ggccaggaac gccagcgtcc ccaccacgac aatacggcgc cacgtcgatt tgctcgttgg 1140ggcggctgct ttctgctccg ctatgtacgt gggggatctc tgcggatctg ttttcctcgt 1200ctcccagttg ttcaccttct cgcctcgccg gcatgagaca gtgcaggact gcaattgttc 1260aatctatccc ggccacgtat caggtcaccg catggcttgg gatatgatga tgaactggtc 1320aactacaaca gccctactgg tatcgcagtt actccggatc ccacaagccg tcgtggacat 1380ggtggcgggg gcccactggg gagtcctggc gggccttgcc tactattcca tggcggggaa 1440ctgggctaag gttttgattg tgctgctact ctttgccggc gttgatgggg cgacctacgt 1500gacggggggg tcggaagcca gaggggcctc tggcttagca aacctctttt catttggggc 1560gtctcagaag atccagctca taaataccaa cggcagttgg cacatcaata gaactgccct 1620gaactgcaat gactccctcc acactgggtt tcttgccgcg ctattctaca cacacaaatt 1680caacgcgtcc ggatgtccag agcgcatggc cagctgccgc cccattgaag agttcgctca 1740ggggtatggt cccatcactt atgctgagcc ctccccctcg gaccagaggc cctattgctg 1800gcactacgcg cctcgaccgt gtggtatcat acccgcgtcg caggtgtgtg gtccagtgta 1860ctgcttcacc ccaagccctg ttgtggtggg gacgaccgat cgctccggtg cccccacgta 1920taattggggg gcgaatgaga cggacgtgct gtatctcaac aacacgcggc cgccgcaagg 1980caactggttc ggctgcacat ggatgaatgg caccgggttc accaagacgt gcgggggccc 2040cccgtgcaac atcggggggg gcggcaacaa caacaccttg acctgcccca cggactgttt 2100ccggaaacac cccgaggcca cctacaccaa atgtggttcg ggaccttggt tgacacctag 2160gtgcatggtc gactacccat acaggctctg gcactacccc tgcaccgtta actttaccat 2220ctttaaggtt aggatgtacg tgggaggtgt ggagcacagg ctcaacgccg catgcaattg 2280gacccgagga gagcgttgta acttagagga cagggataga tcagagctta gcccgctgct 2340gctgtcaaca acagagtggc aggtgctacc ttgttccttc accaccctac cggctctgtc 2400cactggtttg atccatctcc accagaacat cgtggacgtg caatacctgt acggtatagg 2460gtcggcggtt gtctcctatg caatcaaatg ggaatatgtc ttgttgctct tcctcctcct 2520ggcagacgcg cgcgtctgcg cctgcttgtg gatgatgctg ctgatagctc aagctgaggc 2580cgccttagag aacctggtgg tcctcaatgc ggcgtccctg gctggagcgc atggccttct 2640ctctttcctt gtgttcttct gtgccgcttg gtacatcaag ggcaggttga tccccggggc 2700ggcgtatgct ttttacggcg tatggccgct gctcctactc ctgctggcgt taccaccacg 2760agcatacgcc atggaccggg agatggctgc atcgtgcgga ggcgcggttt ttgtaggtct 2820ggcattcctg accttgtcac cacactataa ggcattcctc gccaagctcc tgtggtggtt 2880gtgctatctc ctgaccctgg gggaagccat gattcaggag tgggtaccac ccatgcaggt 2940gcgcggcggc cgcgatggca tcgcgtgggc cgtcactata ttctgcccgg gtgtggtgtt 3000tgacattacc aaatggcttt tggcgttgct tgggcctgct tacctcttaa gggccgcttt 3060gacacatgtg ccgtacttcg tcagagctca cgctctgata agggtatgcg ctttggtgaa 3120gcagctcgcg gggggtaggt atgttcaggt ggcgctattg gcccttggca ggtggactgg 3180cacctacatc tatgaccacc tcacacctat gtcggactgg gccgctagcg gcctgcgcga 3240cttagcggtc gccgtggaac ccatcatctt cagtccgatg gagaagaagg tcatcgtctg 3300gggagcggag acggctgcat gtggggacat tctacatgga cttcccgtgt ccgcccgact 3360cggccaggag atcctcctcg gcccagctga tggctacacc tccaaggggt ggaagctcct 3420tgctcccatc actgcttatg cccagcaaac acgaggcctc ctgggcgcca tagtggtgag 3480tatgacgggg cgtgacagga cagaacaggc cggggaagtc caaatcctgt ccacagtctc 3540tcagtccttc ctcggaacaa ccatctcggg ggttttgtgg actgtttacc acggagctgg 3600caacaagact ctagccggct tacggggtcc ggtcacgcag atgtactcga gtgctgaggg 3660ggacttggta ggctggccca gcccccctgg gaccaagtct ttggagccgt gcaagtgtgg 3720agccgtcgac ctatatctgg tcacgcggaa cgctgatgtc atcccggctc ggagacgcgg 3780ggacaagcgg ggagcattgc tctccccgag acccatttcg accttgaagg ggtcctcggg 3840ggggccggtg ctctgcccta ggggccacgt cgttgggctc ttccgagcag ctgtgtgctc 3900tcggggcgtg gccaaatcca tcgatttcat ccccgttgag acactcgacg ttgttacaag 3960gtctcccact ttcagtgaca acagcacgcc accggctgtg ccccagacct atcaggtcgg 4020gtacttgcat gctccaactg gcagtggaaa gagcaccaag gtccctgtcg cgtatgccgc 4080ccaggggtac aaagtactag tgcttaaccc ctcggtagct gccaccctgg ggtttggggc 4140gtacctatcc aaggcacatg gcatcaatcc caacattagg actggagtca ggaccgtgat 4200gaccggggag gccatcacgt actccacata tggcaaattt ctcgccgatg ggggctgcgc 4260tagcggcgcc tatgacatca tcatatgcga tgaatgccac gctgtggatg ctacctccat 4320tctcggcatc ggaacggtcc ttgatcaagc agagacagcc ggggtcagac taactgtgct 4380ggctacggcc acaccccccg ggtcagtgac aaccccccat cccgatatag aagaggtagg 4440cctcgggcgg gagggtgaga tccccttcta tgggagggcg attcccctat cctgcatcaa 4500gggagggaga cacctgattt tctgccactc aaagaaaaag tgtgacgagc tcgcggcggc 4560ccttcggggc atgggcttga atgccgtggc atactataga gggttggacg tctccataat 4620accagctcag ggagatgtgg tggtcgtcgc caccgacgcc ctcatgacgg ggtacactgg 4680agactttgac tccgtgatcg actgcaatgt agcggtcacc caagctgtcg acttcagcct 4740ggaccccacc ttcactataa ccacacagac tgtcccacaa gacgctgtct cacgcagtca 4800gcgccgcggg cgcacaggta gaggaagaca gggcacttat aggtatgttt ccactggtga 4860acgagcctca ggaatgtttg acagtgtagt gctttgtgag tgctacgacg caggggctgc 4920gtggtacgat ctcacaccag cggagaccac cgtcaggctt agagcgtatt tcaacacgcc 4980cggcctaccc gtgtgtcaag accatcttga attttgggag gcagttttca ccggcctcac 5040acacatagac gcccacttcc tctcccaaac aaagcaagcg ggggagaact tcgcgtacct 5100agtagcctac caagctacgg tgtgcgccag agccaaggcc cctcccccgt cctgggacgc 5160catgtggaag tgcctggccc gactcaagcc tacgcttgcg ggccccacac ctctcctgta 5220ccgtttgggc cctattacca atgaggtcac cctcacacac cctgggacga agtacatcgc

5280cacatgcatg caagctgacc ttgaggtcat gaccagcacg tgggtcctag ctggaggagt 5340cctggcagcc gtcgccgcat attgcctggc gactggatgc gtttccatca tcggccgctt 5400gcacgtcaac cagcgagtcg tcgttgcgcc ggataaggag gtcctgtatg aggcttttga 5460tgagatggag gaatgcgcct ctagggcggc tctcatcgaa gaggggcagc ggatagccga 5520gatgttgaag tccaagatcc aaggcttgct gcagcaggcc tctaagcagg cccaggacat 5580acaacccgct atgcaggctt catggcccaa agtggaacaa ttttgggcca gacacatgtg 5640gaacttcatt agcggcatcc aatacctcgc aggattgtca acactgccag ggaaccccgc 5700ggtggcttcc atgatggcat tcagtgccgc cctcaccagt ccgttgtcga ccagtaccac 5760catccttctc aacatcatgg gaggctggtt agcgtcccag atcgcaccac ccgcgggggc 5820caccggcttt gtcgtcagtg gcctggtggg ggctgccgtg ggcagcatag gcctgggtaa 5880ggtgctggtg gacatcctgg caggatatgg tgcgggcatt tcgggggccc tcgtcgcatt 5940caagatcatg tctggcgaga agccctctat ggaagatgtc atcaatctac tgcctgggat 6000cctgtctccg ggagccctgg tggtgggggt catctgcgcg gccattctgc gccgccacgt 6060gggaccgggg gagggcgcgg tccaatggat gaacaggctt attgcctttg cttccagagg 6120aaaccacgtc gcccctactc actacgtgac ggagtcggat gcgtcgcagc gtgtgaccca 6180actacttggc tctcttacta taaccagcct actcagaaga ctccacaatt ggataactga 6240ggactgcccc atcccatgct ccggatcctg gctccgcgac gtgtgggact gggtttgcac 6300catcttgaca gacttcaaaa attggctgac ctctaaattg ttccccaagc tgcccggcct 6360ccccttcatc tcttgtcaaa aggggtacaa gggtgtgtgg gccggcactg gcatcatgac 6420cacgcgctgc ccttgcggcg ccaacatctc tggcaatgtc cgcctgggct ctatgaggat 6480cacagggcct aaaacctgca tgaacacctg gcaggggacc tttcctatca attgctacac 6540ggagggccag tgcgcgccga aaccccccac gaactacaag accgccatct ggagggtggc 6600ggcctcggag tacgcggagg tgacgcagca tgggtcgtac tcctatgtaa caggactgac 6660cactgacaat ctgaaaattc cttgccaact accttctcca gagtttttct cctgggtgga 6720cggtgtgcag atccataggt ttgcacccac accaaagccg tttttccggg atgaggtctc 6780gttctgcgtt gggcttaatt cctatgctgt cgggtcccag cttccctgtg aacctgagcc 6840cgacgcagac gtattgaggt ccatgctaac agatccgccc cacatcacgg cggagactgc 6900ggcgcggcgc ttggcacggg gatcacctcc atctgaggcg agctcctcag tgagccagct 6960atcagcaccg tcgctgcggg ccacctgcac cacccacagc aacacctatg acgtggacat 7020ggtcgatgcc aacctgctca tggagggcgg tgtggctcag acagagcctg agtccagggt 7080gcccgttctg gactttctcg agccaatggc cgaggaagag agcgaccttg agccctcaat 7140accatcggag tgcatgctcc ccaggagcgg gtttccacgg gccttaccgg cttgggcacg 7200gcctgactac aacccgccgc tcgtggaatc gtggaggagg ccagattacc aaccgcccac 7260cgttgctggt tgtgctctcc ccccccccaa gaaggccccg acgcctcccc caaggagacg 7320ccggacagtg ggtctgagcg agagcaccat atcagaagcc ctccagcaac tggccatcaa 7380gacctttggc cagcccccct cgagcggtga tgcaggctcg tccacggggg cgggcgccgc 7440cgaatccggc ggtccgacgt cccctggtga gccggccccc tcagagacag gttccgcctc 7500ctctatgccc cccctcgagg gggagcctgg agatccggac ctggagtctg atcaggtaga 7560gcttcaacct cccccccagg gggggggggt agctcccggt tcgggctcgg ggtcttggtc 7620tacttgctcc gaggaggacg ataccaccgt gtgctgctcc atgtcatact cctggaccgg 7680ggctctaata actccctgta gccccgaaga ggaaaagttg ccaatcaacc ctttgagtaa 7740ctcgctgttg cgataccata acaaggtgta ctgtacaaca tcaaagagcg cctcacagag 7800ggctaaaaag gtaacttttg acaggacgca agtgctcgac gcccattatg actcagtctt 7860aaaggacatc aagctagcgg cttccaaggt cagcgcaagg ctcctcacct tggaggaggc 7920gtgccagttg actccacccc attctgcaag atccaagtat ggattcgggg ccaaggaggt 7980ccgcagcttg tccgggaggg ccgttaacca catcaagtcc gtgtggaagg acctcctgga 8040agacccacaa acaccaattc ccacaaccat catggccaaa aatgaggtgt tctgcgtgga 8100ccccgccaag gggggtaaga aaccagctcg cctcatcgtt taccctgacc tcggcgtccg 8160ggtctgcgag aaaatggccc tctatgacat tacacaaaag cttcctcagg cggtaatggg 8220agcttcctat ggcttccagt actcccctgc ccaacgggtg gagtatctct tgaaagcatg 8280ggcggaaaag aaggacccca tgggtttttc gtatgatacc cgatgcttcg actcaaccgt 8340cactgagaga gacatcagga ccgaggagtc catataccag gcctgctccc tgcccgagga 8400ggcccgcact gccatacact cgctgactga gagactttac gtaggagggc ccatgttcaa 8460cagcaagggt caaacctgcg gttacagacg ttgccgcgcc agcggggtgc taaccactag 8520catgggtaac accatcacat gctatgtgaa agccctagcg gcctgcaagg ctgcggggat 8580agttgcgccc acaatgctgg tatgcggcga tgacctagta gtcatctcag aaagccaggg 8640gactgaggag gacgagcgga acctgagagc cttcacggag gccatgacca ggtactctgc 8700ccctcctggt gatcccccca gaccggaata tgacctggag ctaataacat cctgttcctc 8760aaatgtgtct gtggcgttgg gcccgcgggg ccgccgcaga tactacctga ccagagaccc 8820aaccactcca ctcgcccggg ctgcctggga aacagttaga cactccccta tcaattcatg 8880gctgggaaac atcatccagt atgctccaac catatgggtt cgcatggtcc taatgacaca 8940cttcttctcc attctcatgg tccaagacac cctggaccag aacctcaact ttgagatgta 9000tggatcagta tactccgtga atcctttgga ccttccagcc ataattgaga ggttacacgg 9060gcttgacgcc ttttctatgc acacatactc tcaccacgaa ctgacgcggg tggcttcagc 9120cctcagaaaa cttggggcgc cacccctcag ggtgtggaag agtcgggctc gcgcagtcag 9180ggcgtccctc atctcccgtg gagggaaagc ggccgtttgc ggccgatatc tcttcaattg 9240ggcggtgaag accaagctca aactcactcc attgccggag gcgcgcctac tggacttatc 9300cagttggttc accgtcggcg ccggcggggg cgacattttt cacagcgtgt cgcgcgcccg 9360accccgctca ttactcttcg gcctactcct acttttcgta ggggtaggcc tcttcctact 9420ccccgctcgg tagagcggca cacactaggt acactccata gctaactgtt cctttttttt 9480tttttttttt tttttttttt tttttttttt tttttctttt tttttttttt ccctctttct 9540tcccttctca tcttattcta ctttctttct tggtggctcc atcttagccc tagtcacggc 9600tagctgtgaa aggtccgtga gccgcatgac tgcagagagt gccgtaactg gtctctctgc 9660agatcatgt 966939669RNAArtificialpTH/JFH1(PA) 3accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu 60cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc 120cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg 180aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg 240caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg 300cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacga auccuaaacc 360ucaaagaaaa accaaacgua acaccaaccg ccgcccacag gacgucaagu ucccgggcgg 420uggccagauc guugguggag uuuaccuguu gccgcgcagg ggccccaggu ugggugugcg 480cgcgacuagg aagacuuccg agcggucgca accucgugga aggcgacaac cuauccccaa 540ggaucgccga cccgagggca gggccugggc ucagccuggg uacccuuggc cccucuaugg 600caacgagggc augggguggg caggauggcu ccugucaccc cguggcuccc ggccuaguug 660gggccccaau gacccccggc gcaggucgcg 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ccugcuugug gaugaugcug cugauagcuc aagcugaggc 2580cgccuuagag aaccuggugg uccucaaugc ggcgucccug gcuggagcgc auggccuucu 2640cucuuuccuu guguucuucu gugccgcuug guacaucaag ggcagguuga uccccggggc 2700ggcguaugcu uuuuacggcg uauggccgcu gcuccuacuc cugcuggcgu uaccaccacg 2760agcauacgcc auggaccggg agauggcugc aucgugcgga ggcgcgguuu uuguaggucu 2820ggcauuccug accuugucac cacacuauaa ggcauuccuc gccaagcucc uguggugguu 2880gugcuaucuc cugacccugg gggaagccau gauucaggag uggguaccac ccaugcaggu 2940gcgcggcggc cgcgauggca ucgcgugggc cgucacuaua uucugcccgg gugugguguu 3000ugacauuacc aaauggcuuu uggcguugcu ugggccugcu uaccucuuaa gggccgcuuu 3060gacacaugug ccguacuucg ucagagcuca cgcucugaua aggguaugcg cuuuggugaa 3120gcagcucgcg ggggguaggu auguucaggu ggcgcuauug gcccuuggca gguggacugg 3180caccuacauc uaugaccacc ucacaccuau gucggacugg gccgcuagcg gccugcgcga 3240cuuagcgguc gccguggaac ccaucaucuu caguccgaug gagaagaagg ucaucgucug 3300gggagcggag acggcugcau guggggacau ucuacaugga cuucccgugu ccgcccgacu 3360cggccaggag auccuccucg gcccagcuga uggcuacacc uccaaggggu ggaagcuccu 3420ugcucccauc acugcuuaug cccagcaaac acgaggccuc cugggcgcca uaguggugag 3480uaugacgggg cgugacagga cagaacaggc cggggaaguc caaauccugu ccacagucuc 3540ucaguccuuc cucggaacaa ccaucucggg gguuuugugg acuguuuacc acggagcugg 3600caacaagacu cuagccggcu uacggggucc ggucacgcag auguacucga gugcugaggg 3660ggacuuggua ggcuggccca gccccccugg gaccaagucu uuggagccgu gcaagugugg 3720agccgucgac cuauaucugg ucacgcggaa cgcugauguc aucccggcuc ggagacgcgg 3780ggacaagcgg ggagcauugc ucuccccgag acccauuucg accuugaagg gguccucggg 3840ggggccggug cucugcccua ggggccacgu cguugggcuc uuccgagcag cugugugcuc 3900ucggggcgug gccaaaucca ucgauuucau ccccguugag acacucgacg uuguuacaag 3960gucucccacu uucagugaca acagcacgcc accggcugug ccccagaccu aucaggucgg 4020guacuugcau gcuccaacug gcaguggaaa gagcaccaag gucccugucg cguaugccgc 4080ccagggguac aaaguacuag ugcuuaaccc cucgguagcu gccacccugg gguuuggggc 4140guaccuaucc aaggcacaug gcaucaaucc caacauuagg acuggaguca ggaccgugau 4200gaccggggag gccaucacgu acuccacaua uggcaaauuu cucgccgaug ggggcugcgc 4260uagcggcgcc uaugacauca ucauaugcga ugaaugccac gcuguggaug cuaccuccau 4320ucucggcauc ggaacggucc uugaucaagc agagacagcc ggggucagac uaacugugcu 4380ggcuacggcc acaccccccg ggucagugac aaccccccau cccgauauag aagagguagg 4440ccucgggcgg gagggugaga uccccuucua ugggagggcg auuccccuau ccugcaucaa 4500gggagggaga caccugauuu ucugccacuc aaagaaaaag ugugacgagc ucgcggcggc 4560ccuucggggc augggcuuga augccguggc auacuauaga ggguuggacg ucuccauaau 4620accagcucag ggagaugugg uggucgucgc caccgacgcc cucaugacgg gguacacugg 4680agacuuugac uccgugaucg acugcaaugu agcggucacc caagcugucg acuucagccu 4740ggaccccacc uucacuauaa ccacacagac ugucccacaa gacgcugucu cacgcaguca 4800gcgccgcggg cgcacaggua gaggaagaca gggcacuuau agguauguuu ccacugguga 4860acgagccuca ggaauguuug acaguguagu gcuuugugag ugcuacgacg caggggcugc 4920gugguacgau cucacaccag cggagaccac cgucaggcuu agagcguauu ucaacacgcc 4980cggccuaccc gugugucaag accaucuuga auuuugggag gcaguuuuca ccggccucac 5040acacauagac gcccacuucc ucucccaaac aaagcaagcg ggggagaacu ucgcguaccu 5100aguagccuac caagcuacgg ugugcgccag agccaaggcc ccucccccgu ccugggacgc 5160cauguggaag ugccuggccc gacucaagcc uacgcuugcg ggccccacac cucuccugua 5220ccguuugggc ccuauuacca augaggucac ccucacacac ccugggacga aguacaucgc 5280cacaugcaug caagcugacc uugaggucau gaccagcacg uggguccuag cuggaggagu 5340ccuggcagcc gucgccgcau auugccuggc gacuggaugc guuuccauca ucggccgcuu 5400gcacgucaac cagcgagucg ucguugcgcc ggauaaggag guccuguaug aggcuuuuga 5460ugagauggag gaaugcgccu cuagggcggc ucucaucgaa gaggggcagc ggauagccga 5520gauguugaag uccaagaucc aaggcuugcu gcagcaggcc ucuaagcagg cccaggacau 5580acaacccgcu augcaggcuu cauggcccaa aguggaacaa uuuugggcca gacacaugug 5640gaacuucauu agcggcaucc aauaccucgc aggauuguca acacugccag ggaaccccgc 5700gguggcuucc augauggcau ucagugccgc ccucaccagu ccguugucga ccaguaccac 5760cauccuucuc aacaucaugg gaggcugguu agcgucccag aucgcaccac ccgcgggggc 5820caccggcuuu gucgucagug gccugguggg ggcugccgug ggcagcauag gccuggguaa 5880ggugcuggug gacauccugg caggauaugg ugcgggcauu ucgggggccc ucgucgcauu 5940caagaucaug ucuggcgaga agcccucuau ggaagauguc aucaaucuac ugccugggau 6000ccugucuccg ggagcccugg uggugggggu caucugcgcg gccauucugc gccgccacgu 6060gggaccgggg gagggcgcgg uccaauggau gaacaggcuu auugccuuug cuuccagagg 6120aaaccacguc gccccuacuc acuacgugac ggagucggau gcgucgcagc gugugaccca 6180acuacuuggc ucucuuacua uaaccagccu acucagaaga cuccacaauu ggauaacuga 6240ggacugcccc aucccaugcu ccggauccug gcuccgcgac gugugggacu ggguuugcac 6300caucuugaca gacuucaaaa auuggcugac cucuaaauug uuccccaagc ugcccggccu 6360ccccuucauc ucuugucaaa agggguacaa gggugugugg gccggcacug gcaucaugac 6420cacgcgcugc ccuugcggcg ccaacaucuc uggcaauguc cgccugggcu cuaugaggau 6480cacagggccu aaaaccugca ugaacaccug gcaggggacc uuuccuauca auugcuacac 6540ggagggccag ugcgcgccga aaccccccac gaacuacaag accgccaucu ggaggguggc 6600ggccucggag uacgcggagg ugacgcagca ugggucguac uccuauguaa caggacugac 6660cacugacaau cugaaaauuc cuugccaacu accuucucca gaguuuuucu ccugggugga 6720cggugugcag auccauaggu uugcacccac accaaagccg uuuuuccggg augaggucuc 6780guucugcguu gggcuuaauu ccuaugcugu cgggucccag cuucccugug aaccugagcc 6840cgacgcagac guauugaggu ccaugcuaac agauccgccc cacaucacgg cggagacugc 6900ggcgcggcgc uuggcacggg gaucaccucc aucugaggcg agcuccucag ugagccagcu 6960aucagcaccg ucgcugcggg ccaccugcac cacccacagc aacaccuaug acguggacau 7020ggucgaugcc aaccugcuca uggagggcgg uguggcucag acagagccug aguccagggu 7080gcccguucug gacuuucucg agccaauggc cgaggaagag agcgaccuug agcccucaau 7140accaucggag ugcaugcucc ccaggagcgg guuuccacgg gccuuaccgg cuugggcacg 7200gccugacuac aacccgccgc ucguggaauc guggaggagg ccagauuacc aaccgcccac 7260cguugcuggu ugugcucucc ccccccccaa gaaggccccg acgccucccc caaggagacg 7320ccggacagug ggucugagcg agagcaccau aucagaagcc cuccagcaac uggccaucaa 7380gaccuuuggc cagccccccu cgagcgguga ugcaggcucg uccacggggg cgggcgccgc 7440cgaauccggc gguccgacgu ccccugguga gccggccccc ucagagacag guuccgccuc 7500cucuaugccc ccccucgagg gggagccugg agauccggac cuggagucug aucagguaga 7560gcuucaaccu cccccccagg gggggggggu agcucccggu ucgggcucgg ggucuugguc 7620uacuugcucc gaggaggacg auaccaccgu gugcugcucc augucauacu ccuggaccgg 7680ggcucuaaua acucccugua gccccgaaga ggaaaaguug ccaaucaacc cuuugaguaa 7740cucgcuguug cgauaccaua acaaggugua cuguacaaca ucaaagagcg ccucacagag 7800ggcuaaaaag guaacuuuug acaggacgca agugcucgac gcccauuaug acucagucuu 7860aaaggacauc aagcuagcgg cuuccaaggu cagcgcaagg cuccucaccu uggaggaggc 7920gugccaguug acuccacccc auucugcaag auccaaguau ggauucgggg ccaaggaggu 7980ccgcagcuug uccgggaggg ccguuaacca caucaagucc guguggaagg accuccugga 8040agacccacaa acaccaauuc ccacaaccau cauggccaaa aaugaggugu ucugcgugga 8100ccccgccaag ggggguaaga aaccagcucg ccucaucguu uacccugacc ucggcguccg 8160ggucugcgag aaaauggccc ucuaugacau uacacaaaag cuuccucagg cgguaauggg 8220agcuuccuau ggcuuccagu acuccccugc ccaacgggug gaguaucucu ugaaagcaug 8280ggcggaaaag aaggacccca uggguuuuuc guaugauacc cgaugcuucg acucaaccgu 8340cacugagaga gacaucagga ccgaggaguc cauauaccag gccugcuccc ugcccgagga 8400ggcccgcacu gccauacacu cgcugacuga gagacuuuac guaggagggc ccauguucaa 8460cagcaagggu caaaccugcg guuacagacg uugccgcgcc agcggggugc uaaccacuag 8520cauggguaac accaucacau gcuaugugaa agcccuagcg gccugcaagg cugcggggau 8580aguugcgccc acaaugcugg uaugcggcga ugaccuagua gucaucucag aaagccaggg 8640gacugaggag gacgagcgga accugagagc cuucacggag gccaugacca gguacucugc 8700cccuccuggu gaucccccca gaccggaaua ugaccuggag cuaauaacau ccuguuccuc 8760aaaugugucu guggcguugg gcccgcgggg ccgccgcaga uacuaccuga ccagagaccc 8820aaccacucca cucgcccggg cugccuggga aacaguuaga cacuccccua ucaauucaug 8880gcugggaaac aucauccagu augcuccaac cauauggguu cgcauggucc uaaugacaca 8940cuucuucucc auucucaugg uccaagacac ccuggaccag aaccucaacu uugagaugua 9000uggaucagua uacuccguga auccuuugga ccuuccagcc auaauugaga gguuacacgg 9060gcuugacgcc uuuucuaugc acacauacuc ucaccacgaa cugacgcggg uggcuucagc 9120ccucagaaaa cuuggggcgc caccccucag gguguggaag agucgggcuc gcgcagucag 9180ggcgucccuc aucucccgug gagggaaagc ggccguuugc ggccgauauc ucuucaauug 9240ggcggugaag accaagcuca aacucacucc auugccggag gcgcgccuac uggacuuauc 9300caguugguuc accgucggcg ccggcggggg cgacauuuuu cacagcgugu cgcgcgcccg 9360accccgcuca uuacucuucg gccuacuccu acuuuucgua gggguaggcc ucuuccuacu 9420ccccgcucgg uagagcggca cacacuaggu acacuccaua gcuaacuguu ccuuuuuuuu 9480uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuucuuuu uuuuuuuuuu cccucuuucu 9540ucccuucuca ucuuauucua cuuucuuucu ugguggcucc aucuuagccc uagucacggc 9600uagcugugaa agguccguga gccgcaugac ugcagagagu gccguaacug gucucucugc 9660agaucaugu 966953030PRTArtificialTH/JFH-1 5Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn1 5 10 15Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 55 60Ile Pro Lys Asp Arg Arg Pro Glu Gly Arg Ala Trp Ala Gln Pro Gly65 70 75 80Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala Gly Trp 85 90 95Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Asn Asp Pro 100 105 110Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 115 120 125Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu 130 135 140Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp145 150 155 160Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile 165 170 175Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr 180 185 190Glu Val Arg Asn Val Ser Gly Val Tyr His Val Thr Asn Asp Cys Ser 195 200 205Asn Ser Ser Ile Val Tyr Glu Thr Gly Asp Met Ile Met His Thr Pro 210 215 220Gly Cys Val Pro Cys Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Ala225 230 235 240Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr 245 250 255Thr Ile Arg Arg His Val Asp Leu Leu Val Gly Ala Ala Ala Phe Cys 260 265 270Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser 275 280 285Gln Leu Phe Thr Phe Ser Pro Arg Arg His Glu Thr Val Gln Asp Cys 290 295 300Asn Cys Ser Ile Tyr Pro Gly His Val Ser Gly His Arg Met Ala Trp305 310 315 320Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Leu Val Ser Gln 325 330 335Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala His 340 345 350Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Ala Gly Asn Trp 355 360 365Ala Lys Val Leu Ile Val Leu Leu Leu Phe Ala Gly Val Asp Gly Ala 370 375 380Thr Tyr Val Thr Gly Gly Ser Glu Ala Arg Gly Ala Ser Gly Leu Ala385 390 395 400Asn Leu Phe Ser Phe Gly Ala Ser Gln Lys Ile Gln Leu Ile Asn Thr 405 410 415Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser 420 425 430Leu His Thr Gly Phe Leu Ala Ala Leu Phe Tyr Thr His Lys Phe Asn 435 440 445Ala Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg Pro Ile Glu Glu 450 455 460Phe Ala Gln Gly Tyr Gly Pro Ile Thr Tyr Ala Glu Pro Ser Pro Ser465 470 475 480Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile 485 490 495Ile Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser 500 505 510Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Asn 515 520 525Trp Gly Ala Asn Glu Thr Asp Val Leu Tyr Leu Asn Asn Thr Arg Pro 530 535 540Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr Gly Phe545 550 555 560Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Gly Gly Asn 565 570 575Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu 580 585 590Ala Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys 595 600 605Met Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn 610 615 620Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg625 630 635 640Leu Asn Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asn Leu Glu 645 650 655Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu 660 665 670Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr 675 680 685Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr 690 695 700Gly Ile Gly Ser Ala Val Val Ser Tyr Ala Ile Lys Trp Glu Tyr Val705 710 715 720Leu Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ala Cys Leu 725 730 735Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu 740 745 750Val Val Leu Asn Ala Ala Ser Leu Ala Gly Ala His Gly Leu Leu Ser 755 760 765Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Ile 770 775 780Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro Leu Leu Leu Leu785 790 795 800Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala 805 810 815Ala Ser Cys Gly Gly Ala Val Phe Val Gly Leu Ala Phe Leu Thr Leu 820 825 830Ser Pro His Tyr Lys Ala Phe Leu Ala Lys Leu Leu Trp Trp Leu Cys 835 840 845Tyr Leu Leu Thr Leu Gly Glu Ala Met Ile Gln Glu Trp Val Pro Pro 850 855 860Met Gln Val Arg Gly Gly Arg Asp Gly Ile Ala Trp Ala Val Thr Ile865 870 875 880Phe Cys Pro Gly Val Val Phe Asp Ile Thr Lys Trp Leu Leu Ala Leu 885 890 895Leu Gly Pro Ala Tyr Leu Leu Arg Ala Ala Leu Thr His Val Pro Tyr 900 905 910Phe Val Arg Ala His Ala Leu Ile Arg Val Cys Ala Leu Val Lys Gln 915 920 925Leu Ala Gly Gly Arg Tyr Val Gln Val Ala Leu Leu Ala Leu Gly Arg 930 935 940Trp Thr Gly Thr Tyr Ile Tyr Asp His Leu Thr Pro Met Ser Asp Trp945 950 955 960Ala Ala Ser Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Ile Ile 965 970 975Phe Ser Pro Met Glu Lys Lys Val Ile Val Trp Gly Ala Glu Thr Ala 980 985 990Ala Cys Gly Asp Ile Leu His Gly Leu Pro Val Ser Ala Arg Leu Gly 995 1000 1005Gln Glu Ile

Leu Leu Gly Pro Ala Asp Gly Tyr Thr Ser Lys Gly 1010 1015 1020Trp Lys Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg 1025 1030 1035Gly Leu Leu Gly Ala Ile Val Val Ser Met Thr Gly Arg Asp Arg 1040 1045 1050Thr Glu Gln Ala Gly Glu Val Gln Ile Leu Ser Thr Val Ser Gln 1055 1060 1065Ser Phe Leu Gly Thr Thr Ile Ser Gly Val Leu Trp Thr Val Tyr 1070 1075 1080His Gly Ala Gly Asn Lys Thr Leu Ala Gly Leu Arg Gly Pro Val 1085 1090 1095Thr Gln Met Tyr Ser Ser Ala Glu Gly Asp Leu Val Gly Trp Pro 1100 1105 1110Ser Pro Pro Gly Thr Lys Ser Leu Glu Pro Cys Lys Cys Gly Ala 1115 1120 1125Val Asp Leu Tyr Leu Val Thr Arg Asn Ala Asp Val Ile Pro Ala 1130 1135 1140Arg Arg Arg Gly Asp Lys Arg Gly Ala Leu Leu Ser Pro Arg Pro 1145 1150 1155Ile Ser Thr Leu Lys Gly Ser Ser Gly Gly Pro Val Leu Cys Pro 1160 1165 1170Arg Gly His Val Val Gly Leu Phe Arg Ala Ala Val Cys Ser Arg 1175 1180 1185Gly Val Ala Lys Ser Ile Asp Phe Ile Pro Val Glu Thr Leu Asp 1190 1195 1200Val Val Thr Arg Ser Pro Thr Phe Ser Asp Asn Ser Thr Pro Pro 1205 1210 1215Ala Val Pro Gln Thr Tyr Gln Val Gly Tyr Leu His Ala Pro Thr 1220 1225 1230Gly Ser Gly Lys Ser Thr Lys Val Pro Val Ala Tyr Ala Ala Gln 1235 1240 1245Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu 1250 1255 1260Gly Phe Gly Ala Tyr Leu Ser Lys Ala His Gly Ile Asn Pro Asn 1265 1270 1275Ile Arg Thr Gly Val Arg Thr Val Met Thr Gly Glu Ala Ile Thr 1280 1285 1290Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ala Ser 1295 1300 1305Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ala Val Asp 1310 1315 1320Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu 1325 1330 1335Thr Ala Gly Val Arg Leu Thr Val Leu Ala Thr Ala Thr Pro Pro 1340 1345 1350Gly Ser Val Thr Thr Pro His Pro Asp Ile Glu Glu Val Gly Leu 1355 1360 1365Gly Arg Glu Gly Glu Ile Pro Phe Tyr Gly Arg Ala Ile Pro Leu 1370 1375 1380Ser Cys Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys 1385 1390 1395Lys Lys Cys Asp Glu Leu Ala Ala Ala Leu Arg Gly Met Gly Leu 1400 1405 1410Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Ile Ile Pro 1415 1420 1425Ala Gln Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr 1430 1435 1440Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Val Ala 1445 1450 1455Val Thr Gln Ala Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile 1460 1465 1470Thr Thr Gln Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg 1475 1480 1485Arg Gly Arg Thr Gly Arg Gly Arg Gln Gly Thr Tyr Arg Tyr Val 1490 1495 1500Ser Thr Gly Glu Arg Ala Ser Gly Met Phe Asp Ser Val Val Leu 1505 1510 1515Cys Glu Cys Tyr Asp Ala Gly Ala Ala Trp Tyr Asp Leu Thr Pro 1520 1525 1530Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Phe Asn Thr Pro Gly 1535 1540 1545Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ala Val Phe 1550 1555 1560Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys 1565 1570 1575Gln Ala Gly Glu Asn Phe Ala Tyr Leu Val Ala Tyr Gln Ala Thr 1580 1585 1590Val Cys Ala Arg Ala Lys Ala Pro Pro Pro Ser Trp Asp Ala Met 1595 1600 1605Trp Lys Cys Leu Ala Arg Leu Lys Pro Thr Leu Ala Gly Pro Thr 1610 1615 1620Pro Leu Leu Tyr Arg Leu Gly Pro Ile Thr Asn Glu Val Thr Leu 1625 1630 1635Thr His Pro Gly Thr Lys Tyr Ile Ala Thr Cys Met Gln Ala Asp 1640 1645 1650Leu Glu Val Met Thr Ser Thr Trp Val Leu Ala Gly Gly Val Leu 1655 1660 1665Ala Ala Val Ala Ala Tyr Cys Leu Ala Thr Gly Cys Val Ser Ile 1670 1675 1680Ile Gly Arg Leu His Val Asn Gln Arg Val Val Val Ala Pro Asp 1685 1690 1695Lys Glu Val Leu Tyr Glu Ala Phe Asp Glu Met Glu Glu Cys Ala 1700 1705 1710Ser Arg Ala Ala Leu Ile Glu Glu Gly Gln Arg Ile Ala Glu Met 1715 1720 1725Leu Lys Ser Lys Ile Gln Gly Leu Leu Gln Gln Ala Ser Lys Gln 1730 1735 1740Ala Gln Asp Ile Gln Pro Ala Met Gln Ala Ser Trp Pro Lys Val 1745 1750 1755Glu Gln Phe Trp Ala Arg His Met Trp Asn Phe Ile Ser Gly Ile 1760 1765 1770Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Val 1775 1780 1785Ala Ser Met Met Ala Phe Ser Ala Ala Leu Thr Ser Pro Leu Ser 1790 1795 1800Thr Ser Thr Thr Ile Leu Leu Asn Ile Met Gly Gly Trp Leu Ala 1805 1810 1815Ser Gln Ile Ala Pro Pro Ala Gly Ala Thr Gly Phe Val Val Ser 1820 1825 1830Gly Leu Val Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val 1835 1840 1845Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Ile Ser Gly Ala 1850 1855 1860Leu Val Ala Phe Lys Ile Met Ser Gly Glu Lys Pro Ser Met Glu 1865 1870 1875Asp Val Ile Asn Leu Leu Pro Gly Ile Leu Ser Pro Gly Ala Leu 1880 1885 1890Val Val Gly Val Ile Cys Ala Ala Ile Leu Arg Arg His Val Gly 1895 1900 1905Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe 1910 1915 1920Ala Ser Arg Gly Asn His Val Ala Pro Thr His Tyr Val Thr Glu 1925 1930 1935Ser Asp Ala Ser Gln Arg Val Thr Gln Leu Leu Gly Ser Leu Thr 1940 1945 1950Ile Thr Ser Leu Leu Arg Arg Leu His Asn Trp Ile Thr Glu Asp 1955 1960 1965Cys Pro Ile Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp Asp 1970 1975 1980Trp Val Cys Thr Ile Leu Thr Asp Phe Lys Asn Trp Leu Thr Ser 1985 1990 1995Lys Leu Phe Pro Lys Leu Pro Gly Leu Pro Phe Ile Ser Cys Gln 2000 2005 2010Lys Gly Tyr Lys Gly Val Trp Ala Gly Thr Gly Ile Met Thr Thr 2015 2020 2025Arg Cys Pro Cys Gly Ala Asn Ile Ser Gly Asn Val Arg Leu Gly 2030 2035 2040Ser Met Arg Ile Thr Gly Pro Lys Thr Cys Met Asn Thr Trp Gln 2045 2050 2055Gly Thr Phe Pro Ile Asn Cys Tyr Thr Glu Gly Gln Cys Ala Pro 2060 2065 2070Lys Pro Pro Thr Asn Tyr Lys Thr Ala Ile Trp Arg Val Ala Ala 2075 2080 2085Ser Glu Tyr Ala Glu Val Thr Gln His Gly Ser Tyr Ser Tyr Val 2090 2095 2100Thr Gly Leu Thr Thr Asp Asn Leu Lys Ile Pro Cys Gln Leu Pro 2105 2110 2115Ser Pro Glu Phe Phe Ser Trp Val Asp Gly Val Gln Ile His Arg 2120 2125 2130Phe Ala Pro Thr Pro Lys Pro Phe Phe Arg Asp Glu Val Ser Phe 2135 2140 2145Cys Val Gly Leu Asn Ser Tyr Ala Val Gly Ser Gln Leu Pro Cys 2150 2155 2160Glu Pro Glu Pro Asp Ala Asp Val Leu Arg Ser Met Leu Thr Asp 2165 2170 2175Pro Pro His Ile Thr Ala Glu Thr Ala Ala Arg Arg Leu Ala Arg 2180 2185 2190Gly Ser Pro Pro Ser Glu Ala Ser Ser Ser Val Ser Gln Leu Ser 2195 2200 2205Ala Pro Ser Leu Arg Ala Thr Cys Thr Thr His Ser Asn Thr Tyr 2210 2215 2220Asp Val Asp Met Val Asp Ala Asn Leu Leu Met Glu Gly Gly Val 2225 2230 2235Ala Gln Thr Glu Pro Glu Ser Arg Val Pro Val Leu Asp Phe Leu 2240 2245 2250Glu Pro Met Ala Glu Glu Glu Ser Asp Leu Glu Pro Ser Ile Pro 2255 2260 2265Ser Glu Cys Met Leu Pro Arg Ser Gly Phe Pro Arg Ala Leu Pro 2270 2275 2280Ala Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val Glu Ser Trp 2285 2290 2295Arg Arg Pro Asp Tyr Gln Pro Pro Thr Val Ala Gly Cys Ala Leu 2300 2305 2310Pro Pro Pro Lys Lys Ala Pro Thr Pro Pro Pro Arg Arg Arg Arg 2315 2320 2325Thr Val Gly Leu Ser Glu Ser Thr Ile Ser Glu Ala Leu Gln Gln 2330 2335 2340Leu Ala Ile Lys Thr Phe Gly Gln Pro Pro Ser Ser Gly Asp Ala 2345 2350 2355Gly Ser Ser Thr Gly Ala Gly Ala Ala Glu Ser Gly Gly Pro Thr 2360 2365 2370Ser Pro Gly Glu Pro Ala Pro Ser Glu Thr Gly Ser Ala Ser Ser 2375 2380 2385Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Glu Ser 2390 2395 2400Asp Gln Val Glu Leu Gln Pro Pro Pro Gln Gly Gly Gly Val Ala 2405 2410 2415Pro Gly Ser Gly Ser Gly Ser Trp Ser Thr Cys Ser Glu Glu Asp 2420 2425 2430Asp Thr Thr Val Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala 2435 2440 2445Leu Ile Thr Pro Cys Ser Pro Glu Glu Glu Lys Leu Pro Ile Asn 2450 2455 2460Pro Leu Ser Asn Ser Leu Leu Arg Tyr His Asn Lys Val Tyr Cys 2465 2470 2475Thr Thr Ser Lys Ser Ala Ser Gln Arg Ala Lys Lys Val Thr Phe 2480 2485 2490Asp Arg Thr Gln Val Leu Asp Ala His Tyr Asp Ser Val Leu Lys 2495 2500 2505Asp Ile Lys Leu Ala Ala Ser Lys Val Ser Ala Arg Leu Leu Thr 2510 2515 2520Leu Glu Glu Ala Cys Gln Leu Thr Pro Pro His Ser Ala Arg Ser 2525 2530 2535Lys Tyr Gly Phe Gly Ala Lys Glu Val Arg Ser Leu Ser Gly Arg 2540 2545 2550Ala Val Asn His Ile Lys Ser Val Trp Lys Asp Leu Leu Glu Asp 2555 2560 2565Pro Gln Thr Pro Ile Pro Thr Thr Ile Met Ala Lys Asn Glu Val 2570 2575 2580Phe Cys Val Asp Pro Ala Lys Gly Gly Lys Lys Pro Ala Arg Leu 2585 2590 2595Ile Val Tyr Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala 2600 2605 2610Leu Tyr Asp Ile Thr Gln Lys Leu Pro Gln Ala Val Met Gly Ala 2615 2620 2625Ser Tyr Gly Phe Gln Tyr Ser Pro Ala Gln Arg Val Glu Tyr Leu 2630 2635 2640Leu Lys Ala Trp Ala Glu Lys Lys Asp Pro Met Gly Phe Ser Tyr 2645 2650 2655Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Arg Asp Ile Arg 2660 2665 2670Thr Glu Glu Ser Ile Tyr Gln Ala Cys Ser Leu Pro Glu Glu Ala 2675 2680 2685Arg Thr Ala Ile His Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly 2690 2695 2700Pro Met Phe Asn Ser Lys Gly Gln Thr Cys Gly Tyr Arg Arg Cys 2705 2710 2715Arg Ala Ser Gly Val Leu Thr Thr Ser Met Gly Asn Thr Ile Thr 2720 2725 2730Cys Tyr Val Lys Ala Leu Ala Ala Cys Lys Ala Ala Gly Ile Val 2735 2740 2745Ala Pro Thr Met Leu Val Cys Gly Asp Asp Leu Val Val Ile Ser 2750 2755 2760Glu Ser Gln Gly Thr Glu Glu Asp Glu Arg Asn Leu Arg Ala Phe 2765 2770 2775Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro 2780 2785 2790Arg Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn 2795 2800 2805Val Ser Val Ala Leu Gly Pro Arg Gly Arg Arg Arg Tyr Tyr Leu 2810 2815 2820Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr 2825 2830 2835Val Arg His Ser Pro Ile Asn Ser Trp Leu Gly Asn Ile Ile Gln 2840 2845 2850Tyr Ala Pro Thr Ile Trp Val Arg Met Val Leu Met Thr His Phe 2855 2860 2865Phe Ser Ile Leu Met Val Gln Asp Thr Leu Asp Gln Asn Leu Asn 2870 2875 2880Phe Glu Met Tyr Gly Ser Val Tyr Ser Val Asn Pro Leu Asp Leu 2885 2890 2895Pro Ala Ile Ile Glu Arg Leu His Gly Leu Asp Ala Phe Ser Met 2900 2905 2910His Thr Tyr Ser His His Glu Leu Thr Arg Val Ala Ser Ala Leu 2915 2920 2925Arg Lys Leu Gly Ala Pro Pro Leu Arg Val Trp Lys Ser Arg Ala 2930 2935 2940Arg Ala Val Arg Ala Ser Leu Ile Ser Arg Gly Gly Lys Ala Ala 2945 2950 2955Val Cys Gly Arg Tyr Leu Phe Asn Trp Ala Val Lys Thr Lys Leu 2960 2965 2970Lys Leu Thr Pro Leu Pro Glu Ala Arg Leu Leu Asp Leu Ser Ser 2975 2980 2985Trp Phe Thr Val Gly Ala Gly Gly Gly Asp Ile Phe His Ser Val 2990 2995 3000Ser Arg Ala Arg Pro Arg Ser Leu Leu Phe Gly Leu Leu Leu Leu 3005 3010 3015Phe Val Gly Val Gly Leu Phe Leu Leu Pro Ala Arg 3020 3025 303063030PRTArtificialTH/JFH-1(PA) 6Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn1 5 10 15Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 55 60Ile Pro Lys Asp Arg Arg Pro Glu Gly Arg Ala Trp Ala Gln Pro Gly65 70 75 80Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala Gly Trp 85 90 95Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Asn Asp Pro 100 105 110Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 115 120 125Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu 130 135 140Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp145 150 155 160Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile 165 170 175Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr 180 185 190Glu Val Arg Asn Val Ser Gly Val Tyr His Val Thr Asn Asp Cys Ser 195 200 205Asn Ser Ser Ile Val Tyr Glu Thr Gly Asp Met Ile Met His Thr Pro 210 215 220Gly Cys Val Pro Cys Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Ala225 230 235 240Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr 245 250 255Thr Ile Arg Arg His Val Asp Leu Leu Val Gly Ala Ala Ala Phe Cys 260 265 270Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser 275 280 285Gln Leu Phe Thr Phe Ser Pro Arg Arg His Glu Thr Val Gln Asp Cys 290 295 300Asn Cys Ser Ile Tyr Pro Gly His Val Ser Gly His Arg Met Ala Trp305 310 315 320Asp Met Met Met Asn Trp Ser Ala Thr Thr Ala Leu Leu Val Ser Gln 325 330 335Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala His 340 345 350Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Ala Gly Asn Trp 355 360 365Ala Lys Val Leu Ile Val Leu Leu Leu Phe Ala Gly Val Asp Gly Ala 370 375 380Thr Tyr Val Thr Gly Gly Ser Glu Ala Arg Gly Ala Ser Gly Leu Ala385 390 395 400Asn Leu Phe Ser Phe Gly Ala Ser Gln Lys Ile Gln Leu Ile Asn Thr 405 410 415Asn Gly Ser

Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser 420 425 430Leu His Thr Gly Phe Leu Ala Ala Leu Phe Tyr Thr His Lys Phe Asn 435 440 445Ala Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg Pro Ile Glu Glu 450 455 460Phe Ala Gln Gly Tyr Gly Pro Ile Thr Tyr Ala Glu Pro Ser Pro Ser465 470 475 480Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile 485 490 495Ile Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser 500 505 510Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Asn 515 520 525Trp Gly Ala Asn Glu Thr Asp Val Leu Tyr Leu Asn Asn Thr Arg Pro 530 535 540Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr Gly Phe545 550 555 560Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Gly Gly Asn 565 570 575Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu 580 585 590Ala Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys 595 600 605Met Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn 610 615 620Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg625 630 635 640Leu Asn Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asn Leu Glu 645 650 655Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu 660 665 670Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr 675 680 685Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr 690 695 700Gly Ile Gly Ser Ala Val Val Ser Tyr Ala Ile Lys Trp Glu Tyr Val705 710 715 720Leu Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ala Cys Leu 725 730 735Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu 740 745 750Val Val Leu Asn Ala Ala Ser Leu Ala Gly Ala His Gly Leu Leu Ser 755 760 765Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Ile 770 775 780Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro Leu Leu Leu Leu785 790 795 800Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala 805 810 815Ala Ser Cys Gly Gly Ala Val Phe Val Gly Leu Ala Phe Leu Thr Leu 820 825 830Ser Pro His Tyr Lys Ala Phe Leu Ala Lys Leu Leu Trp Trp Leu Cys 835 840 845Tyr Leu Leu Thr Leu Gly Glu Ala Met Ile Gln Glu Trp Val Pro Pro 850 855 860Met Gln Val Arg Gly Gly Arg Asp Gly Ile Ala Trp Ala Val Thr Ile865 870 875 880Phe Cys Pro Gly Val Val Phe Asp Ile Thr Lys Trp Leu Leu Ala Leu 885 890 895Leu Gly Pro Ala Tyr Leu Leu Arg Ala Ala Leu Thr His Val Pro Tyr 900 905 910Phe Val Arg Ala His Ala Leu Ile Arg Val Cys Ala Leu Val Lys Gln 915 920 925Leu Ala Gly Gly Arg Tyr Val Gln Val Ala Leu Leu Ala Leu Gly Arg 930 935 940Trp Thr Gly Thr Tyr Ile Tyr Asp His Leu Thr Pro Met Ser Asp Trp945 950 955 960Ala Ala Ser Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Ile Ile 965 970 975Phe Ser Pro Met Glu Lys Lys Val Ile Val Trp Gly Ala Glu Thr Ala 980 985 990Ala Cys Gly Asp Ile Leu His Gly Leu Pro Val Ser Ala Arg Leu Gly 995 1000 1005Gln Glu Ile Leu Leu Gly Pro Ala Asp Gly Tyr Thr Ser Lys Gly 1010 1015 1020Trp Lys Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg 1025 1030 1035Gly Leu Leu Gly Ala Ile Val Val Ser Met Thr Gly Arg Asp Arg 1040 1045 1050Thr Glu Gln Ala Gly Glu Val Gln Ile Leu Ser Thr Val Ser Gln 1055 1060 1065Ser Phe Leu Gly Thr Thr Ile Ser Gly Val Leu Trp Thr Val Tyr 1070 1075 1080His Gly Ala Gly Asn Lys Thr Leu Ala Gly Leu Arg Gly Pro Val 1085 1090 1095Thr Gln Met Tyr Ser Ser Ala Glu Gly Asp Leu Val Gly Trp Pro 1100 1105 1110Ser Pro Pro Gly Thr Lys Ser Leu Glu Pro Cys Lys Cys Gly Ala 1115 1120 1125Val Asp Leu Tyr Leu Val Thr Arg Asn Ala Asp Val Ile Pro Ala 1130 1135 1140Arg Arg Arg Gly Asp Lys Arg Gly Ala Leu Leu Ser Pro Arg Pro 1145 1150 1155Ile Ser Thr Leu Lys Gly Ser Ser Gly Gly Pro Val Leu Cys Pro 1160 1165 1170Arg Gly His Val Val Gly Leu Phe Arg Ala Ala Val Cys Ser Arg 1175 1180 1185Gly Val Ala Lys Ser Ile Asp Phe Ile Pro Val Glu Thr Leu Asp 1190 1195 1200Val Val Thr Arg Ser Pro Thr Phe Ser Asp Asn Ser Thr Pro Pro 1205 1210 1215Ala Val Pro Gln Thr Tyr Gln Val Gly Tyr Leu His Ala Pro Thr 1220 1225 1230Gly Ser Gly Lys Ser Thr Lys Val Pro Val Ala Tyr Ala Ala Gln 1235 1240 1245Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu 1250 1255 1260Gly Phe Gly Ala Tyr Leu Ser Lys Ala His Gly Ile Asn Pro Asn 1265 1270 1275Ile Arg Thr Gly Val Arg Thr Val Met Thr Gly Glu Ala Ile Thr 1280 1285 1290Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ala Ser 1295 1300 1305Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ala Val Asp 1310 1315 1320Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu 1325 1330 1335Thr Ala Gly Val Arg Leu Thr Val Leu Ala Thr Ala Thr Pro Pro 1340 1345 1350Gly Ser Val Thr Thr Pro His Pro Asp Ile Glu Glu Val Gly Leu 1355 1360 1365Gly Arg Glu Gly Glu Ile Pro Phe Tyr Gly Arg Ala Ile Pro Leu 1370 1375 1380Ser Cys Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys 1385 1390 1395Lys Lys Cys Asp Glu Leu Ala Ala Ala Leu Arg Gly Met Gly Leu 1400 1405 1410Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Ile Ile Pro 1415 1420 1425Ala Gln Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr 1430 1435 1440Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Val Ala 1445 1450 1455Val Thr Gln Ala Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile 1460 1465 1470Thr Thr Gln Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg 1475 1480 1485Arg Gly Arg Thr Gly Arg Gly Arg Gln Gly Thr Tyr Arg Tyr Val 1490 1495 1500Ser Thr Gly Glu Arg Ala Ser Gly Met Phe Asp Ser Val Val Leu 1505 1510 1515Cys Glu Cys Tyr Asp Ala Gly Ala Ala Trp Tyr Asp Leu Thr Pro 1520 1525 1530Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Phe Asn Thr Pro Gly 1535 1540 1545Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ala Val Phe 1550 1555 1560Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys 1565 1570 1575Gln Ala Gly Glu Asn Phe Ala Tyr Leu Val Ala Tyr Gln Ala Thr 1580 1585 1590Val Cys Ala Arg Ala Lys Ala Pro Pro Pro Ser Trp Asp Ala Met 1595 1600 1605Trp Lys Cys Leu Ala Arg Leu Lys Pro Thr Leu Ala Gly Pro Thr 1610 1615 1620Pro Leu Leu Tyr Arg Leu Gly Pro Ile Thr Asn Glu Val Thr Leu 1625 1630 1635Thr His Pro Gly Thr Lys Tyr Ile Ala Thr Cys Met Gln Ala Asp 1640 1645 1650Leu Glu Val Met Thr Ser Thr Trp Val Leu Ala Gly Gly Val Leu 1655 1660 1665Ala Ala Val Ala Ala Tyr Cys Leu Ala Thr Gly Cys Val Ser Ile 1670 1675 1680Ile Gly Arg Leu His Val Asn Gln Arg Val Val Val Ala Pro Asp 1685 1690 1695Lys Glu Val Leu Tyr Glu Ala Phe Asp Glu Met Glu Glu Cys Ala 1700 1705 1710Ser Arg Ala Ala Leu Ile Glu Glu Gly Gln Arg Ile Ala Glu Met 1715 1720 1725Leu Lys Ser Lys Ile Gln Gly Leu Leu Gln Gln Ala Ser Lys Gln 1730 1735 1740Ala Gln Asp Ile Gln Pro Ala Met Gln Ala Ser Trp Pro Lys Val 1745 1750 1755Glu Gln Phe Trp Ala Arg His Met Trp Asn Phe Ile Ser Gly Ile 1760 1765 1770Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Val 1775 1780 1785Ala Ser Met Met Ala Phe Ser Ala Ala Leu Thr Ser Pro Leu Ser 1790 1795 1800Thr Ser Thr Thr Ile Leu Leu Asn Ile Met Gly Gly Trp Leu Ala 1805 1810 1815Ser Gln Ile Ala Pro Pro Ala Gly Ala Thr Gly Phe Val Val Ser 1820 1825 1830Gly Leu Val Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val 1835 1840 1845Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Ile Ser Gly Ala 1850 1855 1860Leu Val Ala Phe Lys Ile Met Ser Gly Glu Lys Pro Ser Met Glu 1865 1870 1875Asp Val Ile Asn Leu Leu Pro Gly Ile Leu Ser Pro Gly Ala Leu 1880 1885 1890Val Val Gly Val Ile Cys Ala Ala Ile Leu Arg Arg His Val Gly 1895 1900 1905Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe 1910 1915 1920Ala Ser Arg Gly Asn His Val Ala Pro Thr His Tyr Val Thr Glu 1925 1930 1935Ser Asp Ala Ser Gln Arg Val Thr Gln Leu Leu Gly Ser Leu Thr 1940 1945 1950Ile Thr Ser Leu Leu Arg Arg Leu His Asn Trp Ile Thr Glu Asp 1955 1960 1965Cys Pro Ile Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp Asp 1970 1975 1980Trp Val Cys Thr Ile Leu Thr Asp Phe Lys Asn Trp Leu Thr Ser 1985 1990 1995Lys Leu Phe Pro Lys Leu Pro Gly Leu Pro Phe Ile Ser Cys Gln 2000 2005 2010Lys Gly Tyr Lys Gly Val Trp Ala Gly Thr Gly Ile Met Thr Thr 2015 2020 2025Arg Cys Pro Cys Gly Ala Asn Ile Ser Gly Asn Val Arg Leu Gly 2030 2035 2040Ser Met Arg Ile Thr Gly Pro Lys Thr Cys Met Asn Thr Trp Gln 2045 2050 2055Gly Thr Phe Pro Ile Asn Cys Tyr Thr Glu Gly Gln Cys Ala Pro 2060 2065 2070Lys Pro Pro Thr Asn Tyr Lys Thr Ala Ile Trp Arg Val Ala Ala 2075 2080 2085Ser Glu Tyr Ala Glu Val Thr Gln His Gly Ser Tyr Ser Tyr Val 2090 2095 2100Thr Gly Leu Thr Thr Asp Asn Leu Lys Ile Pro Cys Gln Leu Pro 2105 2110 2115Ser Pro Glu Phe Phe Ser Trp Val Asp Gly Val Gln Ile His Arg 2120 2125 2130Phe Ala Pro Thr Pro Lys Pro Phe Phe Arg Asp Glu Val Ser Phe 2135 2140 2145Cys Val Gly Leu Asn Ser Tyr Ala Val Gly Ser Gln Leu Pro Cys 2150 2155 2160Glu Pro Glu Pro Asp Ala Asp Val Leu Arg Ser Met Leu Thr Asp 2165 2170 2175Pro Pro His Ile Thr Ala Glu Thr Ala Ala Arg Arg Leu Ala Arg 2180 2185 2190Gly Ser Pro Pro Ser Glu Ala Ser Ser Ser Val Ser Gln Leu Ser 2195 2200 2205Ala Pro Ser Leu Arg Ala Thr Cys Thr Thr His Ser Asn Thr Tyr 2210 2215 2220Asp Val Asp Met Val Asp Ala Asn Leu Leu Met Glu Gly Gly Val 2225 2230 2235Ala Gln Thr Glu Pro Glu Ser Arg Val Pro Val Leu Asp Phe Leu 2240 2245 2250Glu Pro Met Ala Glu Glu Glu Ser Asp Leu Glu Pro Ser Ile Pro 2255 2260 2265Ser Glu Cys Met Leu Pro Arg Ser Gly Phe Pro Arg Ala Leu Pro 2270 2275 2280Ala Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val Glu Ser Trp 2285 2290 2295Arg Arg Pro Asp Tyr Gln Pro Pro Thr Val Ala Gly Cys Ala Leu 2300 2305 2310Pro Pro Pro Lys Lys Ala Pro Thr Pro Pro Pro Arg Arg Arg Arg 2315 2320 2325Thr Val Gly Leu Ser Glu Ser Thr Ile Ser Glu Ala Leu Gln Gln 2330 2335 2340Leu Ala Ile Lys Thr Phe Gly Gln Pro Pro Ser Ser Gly Asp Ala 2345 2350 2355Gly Ser Ser Thr Gly Ala Gly Ala Ala Glu Ser Gly Gly Pro Thr 2360 2365 2370Ser Pro Gly Glu Pro Ala Pro Ser Glu Thr Gly Ser Ala Ser Ser 2375 2380 2385Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Glu Ser 2390 2395 2400Asp Gln Val Glu Leu Gln Pro Pro Pro Gln Gly Gly Gly Val Ala 2405 2410 2415Pro Gly Ser Gly Ser Gly Ser Trp Ser Thr Cys Ser Glu Glu Asp 2420 2425 2430Asp Thr Thr Val Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala 2435 2440 2445Leu Ile Thr Pro Cys Ser Pro Glu Glu Glu Lys Leu Pro Ile Asn 2450 2455 2460Pro Leu Ser Asn Ser Leu Leu Arg Tyr His Asn Lys Val Tyr Cys 2465 2470 2475Thr Thr Ser Lys Ser Ala Ser Gln Arg Ala Lys Lys Val Thr Phe 2480 2485 2490Asp Arg Thr Gln Val Leu Asp Ala His Tyr Asp Ser Val Leu Lys 2495 2500 2505Asp Ile Lys Leu Ala Ala Ser Lys Val Ser Ala Arg Leu Leu Thr 2510 2515 2520Leu Glu Glu Ala Cys Gln Leu Thr Pro Pro His Ser Ala Arg Ser 2525 2530 2535Lys Tyr Gly Phe Gly Ala Lys Glu Val Arg Ser Leu Ser Gly Arg 2540 2545 2550Ala Val Asn His Ile Lys Ser Val Trp Lys Asp Leu Leu Glu Asp 2555 2560 2565Pro Gln Thr Pro Ile Pro Thr Thr Ile Met Ala Lys Asn Glu Val 2570 2575 2580Phe Cys Val Asp Pro Ala Lys Gly Gly Lys Lys Pro Ala Arg Leu 2585 2590 2595Ile Val Tyr Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala 2600 2605 2610Leu Tyr Asp Ile Thr Gln Lys Leu Pro Gln Ala Val Met Gly Ala 2615 2620 2625Ser Tyr Gly Phe Gln Tyr Ser Pro Ala Gln Arg Val Glu Tyr Leu 2630 2635 2640Leu Lys Ala Trp Ala Glu Lys Lys Asp Pro Met Gly Phe Ser Tyr 2645 2650 2655Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Arg Asp Ile Arg 2660 2665 2670Thr Glu Glu Ser Ile Tyr Gln Ala Cys Ser Leu Pro Glu Glu Ala 2675 2680 2685Arg Thr Ala Ile His Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly 2690 2695 2700Pro Met Phe Asn Ser Lys Gly Gln Thr Cys Gly Tyr Arg Arg Cys 2705 2710 2715Arg Ala Ser Gly Val Leu Thr Thr Ser Met Gly Asn Thr Ile Thr 2720 2725 2730Cys Tyr Val Lys Ala Leu Ala Ala Cys Lys Ala Ala Gly Ile Val 2735 2740 2745Ala Pro Thr Met Leu Val Cys Gly Asp Asp Leu Val Val Ile Ser 2750 2755 2760Glu Ser Gln Gly Thr Glu Glu Asp Glu Arg Asn Leu Arg Ala Phe 2765 2770 2775Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro 2780 2785 2790Arg Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn 2795 2800 2805Val Ser Val Ala Leu Gly Pro Arg Gly Arg Arg Arg Tyr Tyr Leu 2810 2815 2820Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr 2825 2830 2835Val Arg His Ser Pro Ile Asn Ser Trp Leu Gly Asn Ile Ile Gln 2840 2845 2850Tyr Ala Pro Thr Ile Trp Val Arg Met Val Leu Met Thr His

Phe 2855 2860 2865Phe Ser Ile Leu Met Val Gln Asp Thr Leu Asp Gln Asn Leu Asn 2870 2875 2880Phe Glu Met Tyr Gly Ser Val Tyr Ser Val Asn Pro Leu Asp Leu 2885 2890 2895Pro Ala Ile Ile Glu Arg Leu His Gly Leu Asp Ala Phe Ser Met 2900 2905 2910His Thr Tyr Ser His His Glu Leu Thr Arg Val Ala Ser Ala Leu 2915 2920 2925Arg Lys Leu Gly Ala Pro Pro Leu Arg Val Trp Lys Ser Arg Ala 2930 2935 2940Arg Ala Val Arg Ala Ser Leu Ile Ser Arg Gly Gly Lys Ala Ala 2945 2950 2955Val Cys Gly Arg Tyr Leu Phe Asn Trp Ala Val Lys Thr Lys Leu 2960 2965 2970Lys Leu Thr Pro Leu Pro Glu Ala Arg Leu Leu Asp Leu Ser Ser 2975 2980 2985Trp Phe Thr Val Gly Ala Gly Gly Gly Asp Ile Phe His Ser Val 2990 2995 3000Ser Arg Ala Arg Pro Arg Ser Leu Leu Phe Gly Leu Leu Leu Leu 3005 3010 3015Phe Val Gly Val Gly Leu Phe Leu Leu Pro Ala Arg 3020 3025 303073030PRTArtificialTH/JFH-1(PT) 7Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn1 5 10 15Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 55 60Ile Pro Lys Asp Arg Arg Pro Glu Gly Arg Ala Trp Ala Gln Pro Gly65 70 75 80Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala Gly Trp 85 90 95Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Asn Asp Pro 100 105 110Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 115 120 125Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu 130 135 140Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp145 150 155 160Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile 165 170 175Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr 180 185 190Glu Val Arg Asn Val Ser Gly Val Tyr His Val Thr Asn Asp Cys Ser 195 200 205Asn Ser Ser Ile Val Tyr Glu Thr Gly Asp Met Ile Met His Thr Pro 210 215 220Gly Cys Val Pro Cys Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Ala225 230 235 240Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr 245 250 255Thr Ile Arg Arg His Val Asp Leu Leu Val Gly Ala Ala Ala Phe Cys 260 265 270Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser 275 280 285Gln Leu Phe Thr Phe Ser Pro Arg Arg His Glu Thr Val Gln Asp Cys 290 295 300Asn Cys Ser Ile Tyr Pro Gly His Val Ser Gly His Arg Met Ala Trp305 310 315 320Asp Met Met Met Asn Trp Ser Thr Thr Thr Ala Leu Leu Val Ser Gln 325 330 335Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala His 340 345 350Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Ala Gly Asn Trp 355 360 365Ala Lys Val Leu Ile Val Leu Leu Leu Phe Ala Gly Val Asp Gly Ala 370 375 380Thr Tyr Val Thr Gly Gly Ser Glu Ala Arg Gly Ala Ser Gly Leu Ala385 390 395 400Asn Leu Phe Ser Phe Gly Ala Ser Gln Lys Ile Gln Leu Ile Asn Thr 405 410 415Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser 420 425 430Leu His Thr Gly Phe Leu Ala Ala Leu Phe Tyr Thr His Lys Phe Asn 435 440 445Ala Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg Pro Ile Glu Glu 450 455 460Phe Ala Gln Gly Tyr Gly Pro Ile Thr Tyr Ala Glu Pro Ser Pro Ser465 470 475 480Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile 485 490 495Ile Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser 500 505 510Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr Tyr Asn 515 520 525Trp Gly Ala Asn Glu Thr Asp Val Leu Tyr Leu Asn Asn Thr Arg Pro 530 535 540Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr Gly Phe545 550 555 560Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Gly Gly Asn 565 570 575Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu 580 585 590Ala Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys 595 600 605Met Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn 610 615 620Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg625 630 635 640Leu Asn Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asn Leu Glu 645 650 655Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu 660 665 670Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr 675 680 685Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr 690 695 700Gly Ile Gly Ser Ala Val Val Ser Tyr Ala Ile Lys Trp Glu Tyr Val705 710 715 720Leu Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys Ala Cys Leu 725 730 735Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu 740 745 750Val Val Leu Asn Ala Ala Ser Leu Ala Gly Ala His Gly Leu Leu Ser 755 760 765Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Ile 770 775 780Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro Leu Leu Leu Leu785 790 795 800Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala Met Asp Arg Glu Met Ala 805 810 815Ala Ser Cys Gly Gly Ala Val Phe Val Gly Leu Ala Phe Leu Thr Leu 820 825 830Ser Pro His Tyr Lys Ala Phe Leu Ala Lys Leu Leu Trp Trp Leu Cys 835 840 845Tyr Leu Leu Thr Leu Gly Glu Ala Met Ile Gln Glu Trp Val Pro Pro 850 855 860Met Gln Val Arg Gly Gly Arg Asp Gly Ile Ala Trp Ala Val Thr Ile865 870 875 880Phe Cys Pro Gly Val Val Phe Asp Ile Thr Lys Trp Leu Leu Ala Leu 885 890 895Leu Gly Pro Ala Tyr Leu Leu Arg Ala Ala Leu Thr His Val Pro Tyr 900 905 910Phe Val Arg Ala His Ala Leu Ile Arg Val Cys Ala Leu Val Lys Gln 915 920 925Leu Ala Gly Gly Arg Tyr Val Gln Val Ala Leu Leu Ala Leu Gly Arg 930 935 940Trp Thr Gly Thr Tyr Ile Tyr Asp His Leu Thr Pro Met Ser Asp Trp945 950 955 960Ala Ala Ser Gly Leu Arg Asp Leu Ala Val Ala Val Glu Pro Ile Ile 965 970 975Phe Ser Pro Met Glu Lys Lys Val Ile Val Trp Gly Ala Glu Thr Ala 980 985 990Ala Cys Gly Asp Ile Leu His Gly Leu Pro Val Ser Ala Arg Leu Gly 995 1000 1005Gln Glu Ile Leu Leu Gly Pro Ala Asp Gly Tyr Thr Ser Lys Gly 1010 1015 1020Trp Lys Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg 1025 1030 1035Gly Leu Leu Gly Ala Ile Val Val Ser Met Thr Gly Arg Asp Arg 1040 1045 1050Thr Glu Gln Ala Gly Glu Val Gln Ile Leu Ser Thr Val Ser Gln 1055 1060 1065Ser Phe Leu Gly Thr Thr Ile Ser Gly Val Leu Trp Thr Val Tyr 1070 1075 1080His Gly Ala Gly Asn Lys Thr Leu Ala Gly Leu Arg Gly Pro Val 1085 1090 1095Thr Gln Met Tyr Ser Ser Ala Glu Gly Asp Leu Val Gly Trp Pro 1100 1105 1110Ser Pro Pro Gly Thr Lys Ser Leu Glu Pro Cys Lys Cys Gly Ala 1115 1120 1125Val Asp Leu Tyr Leu Val Thr Arg Asn Ala Asp Val Ile Pro Ala 1130 1135 1140Arg Arg Arg Gly Asp Lys Arg Gly Ala Leu Leu Ser Pro Arg Pro 1145 1150 1155Ile Ser Thr Leu Lys Gly Ser Ser Gly Gly Pro Val Leu Cys Pro 1160 1165 1170Arg Gly His Val Val Gly Leu Phe Arg Ala Ala Val Cys Ser Arg 1175 1180 1185Gly Val Ala Lys Ser Ile Asp Phe Ile Pro Val Glu Thr Leu Asp 1190 1195 1200Val Val Thr Arg Ser Pro Thr Phe Ser Asp Asn Ser Thr Pro Pro 1205 1210 1215Ala Val Pro Gln Thr Tyr Gln Val Gly Tyr Leu His Ala Pro Thr 1220 1225 1230Gly Ser Gly Lys Ser Thr Lys Val Pro Val Ala Tyr Ala Ala Gln 1235 1240 1245Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu 1250 1255 1260Gly Phe Gly Ala Tyr Leu Ser Lys Ala His Gly Ile Asn Pro Asn 1265 1270 1275Ile Arg Thr Gly Val Arg Thr Val Met Thr Gly Glu Ala Ile Thr 1280 1285 1290Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ala Ser 1295 1300 1305Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ala Val Asp 1310 1315 1320Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu 1325 1330 1335Thr Ala Gly Val Arg Leu Thr Val Leu Ala Thr Ala Thr Pro Pro 1340 1345 1350Gly Ser Val Thr Thr Pro His Pro Asp Ile Glu Glu Val Gly Leu 1355 1360 1365Gly Arg Glu Gly Glu Ile Pro Phe Tyr Gly Arg Ala Ile Pro Leu 1370 1375 1380Ser Cys Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys 1385 1390 1395Lys Lys Cys Asp Glu Leu Ala Ala Ala Leu Arg Gly Met Gly Leu 1400 1405 1410Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Ile Ile Pro 1415 1420 1425Ala Gln Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr 1430 1435 1440Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Val Ala 1445 1450 1455Val Thr Gln Ala Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile 1460 1465 1470Thr Thr Gln Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg 1475 1480 1485Arg Gly Arg Thr Gly Arg Gly Arg Gln Gly Thr Tyr Arg Tyr Val 1490 1495 1500Ser Thr Gly Glu Arg Ala Ser Gly Met Phe Asp Ser Val Val Leu 1505 1510 1515Cys Glu Cys Tyr Asp Ala Gly Ala Ala Trp Tyr Asp Leu Thr Pro 1520 1525 1530Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Phe Asn Thr Pro Gly 1535 1540 1545Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ala Val Phe 1550 1555 1560Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys 1565 1570 1575Gln Ala Gly Glu Asn Phe Ala Tyr Leu Val Ala Tyr Gln Ala Thr 1580 1585 1590Val Cys Ala Arg Ala Lys Ala Pro Pro Pro Ser Trp Asp Ala Met 1595 1600 1605Trp Lys Cys Leu Ala Arg Leu Lys Pro Thr Leu Ala Gly Pro Thr 1610 1615 1620Pro Leu Leu Tyr Arg Leu Gly Pro Ile Thr Asn Glu Val Thr Leu 1625 1630 1635Thr His Pro Gly Thr Lys Tyr Ile Ala Thr Cys Met Gln Ala Asp 1640 1645 1650Leu Glu Val Met Thr Ser Thr Trp Val Leu Ala Gly Gly Val Leu 1655 1660 1665Ala Ala Val Ala Ala Tyr Cys Leu Ala Thr Gly Cys Val Ser Ile 1670 1675 1680Ile Gly Arg Leu His Val Asn Gln Arg Val Val Val Ala Pro Asp 1685 1690 1695Lys Glu Val Leu Tyr Glu Ala Phe Asp Glu Met Glu Glu Cys Ala 1700 1705 1710Ser Arg Ala Ala Leu Ile Glu Glu Gly Gln Arg Ile Ala Glu Met 1715 1720 1725Leu Lys Ser Lys Ile Gln Gly Leu Leu Gln Gln Ala Ser Lys Gln 1730 1735 1740Ala Gln Asp Ile Gln Pro Ala Met Gln Ala Ser Trp Pro Lys Val 1745 1750 1755Glu Gln Phe Trp Ala Arg His Met Trp Asn Phe Ile Ser Gly Ile 1760 1765 1770Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Val 1775 1780 1785Ala Ser Met Met Ala Phe Ser Ala Ala Leu Thr Ser Pro Leu Ser 1790 1795 1800Thr Ser Thr Thr Ile Leu Leu Asn Ile Met Gly Gly Trp Leu Ala 1805 1810 1815Ser Gln Ile Ala Pro Pro Ala Gly Ala Thr Gly Phe Val Val Ser 1820 1825 1830Gly Leu Val Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val 1835 1840 1845Leu Val Asp Ile Leu Ala Gly Tyr Gly Ala Gly Ile Ser Gly Ala 1850 1855 1860Leu Val Ala Phe Lys Ile Met Ser Gly Glu Lys Pro Ser Met Glu 1865 1870 1875Asp Val Ile Asn Leu Leu Pro Gly Ile Leu Ser Pro Gly Ala Leu 1880 1885 1890Val Val Gly Val Ile Cys Ala Ala Ile Leu Arg Arg His Val Gly 1895 1900 1905Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe 1910 1915 1920Ala Ser Arg Gly Asn His Val Ala Pro Thr His Tyr Val Thr Glu 1925 1930 1935Ser Asp Ala Ser Gln Arg Val Thr Gln Leu Leu Gly Ser Leu Thr 1940 1945 1950Ile Thr Ser Leu Leu Arg Arg Leu His Asn Trp Ile Thr Glu Asp 1955 1960 1965Cys Pro Ile Pro Cys Ser Gly Ser Trp Leu Arg Asp Val Trp Asp 1970 1975 1980Trp Val Cys Thr Ile Leu Thr Asp Phe Lys Asn Trp Leu Thr Ser 1985 1990 1995Lys Leu Phe Pro Lys Leu Pro Gly Leu Pro Phe Ile Ser Cys Gln 2000 2005 2010Lys Gly Tyr Lys Gly Val Trp Ala Gly Thr Gly Ile Met Thr Thr 2015 2020 2025Arg Cys Pro Cys Gly Ala Asn Ile Ser Gly Asn Val Arg Leu Gly 2030 2035 2040Ser Met Arg Ile Thr Gly Pro Lys Thr Cys Met Asn Thr Trp Gln 2045 2050 2055Gly Thr Phe Pro Ile Asn Cys Tyr Thr Glu Gly Gln Cys Ala Pro 2060 2065 2070Lys Pro Pro Thr Asn Tyr Lys Thr Ala Ile Trp Arg Val Ala Ala 2075 2080 2085Ser Glu Tyr Ala Glu Val Thr Gln His Gly Ser Tyr Ser Tyr Val 2090 2095 2100Thr Gly Leu Thr Thr Asp Asn Leu Lys Ile Pro Cys Gln Leu Pro 2105 2110 2115Ser Pro Glu Phe Phe Ser Trp Val Asp Gly Val Gln Ile His Arg 2120 2125 2130Phe Ala Pro Thr Pro Lys Pro Phe Phe Arg Asp Glu Val Ser Phe 2135 2140 2145Cys Val Gly Leu Asn Ser Tyr Ala Val Gly Ser Gln Leu Pro Cys 2150 2155 2160Glu Pro Glu Pro Asp Ala Asp Val Leu Arg Ser Met Leu Thr Asp 2165 2170 2175Pro Pro His Ile Thr Ala Glu Thr Ala Ala Arg Arg Leu Ala Arg 2180 2185 2190Gly Ser Pro Pro Ser Glu Ala Ser Ser Ser Val Ser Gln Leu Ser 2195 2200 2205Ala Pro Ser Leu Arg Ala Thr Cys Thr Thr His Ser Asn Thr Tyr 2210 2215 2220Asp Val Asp Met Val Asp Ala Asn Leu Leu Met Glu Gly Gly Val 2225 2230 2235Ala Gln Thr Glu Pro Glu Ser Arg Val Pro Val Leu Asp Phe Leu 2240 2245 2250Glu Pro Met Ala Glu Glu Glu Ser Asp Leu Glu Pro Ser Ile Pro 2255 2260 2265Ser Glu Cys Met Leu Pro Arg Ser Gly Phe Pro Arg Ala Leu Pro

2270 2275 2280Ala Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val Glu Ser Trp 2285 2290 2295Arg Arg Pro Asp Tyr Gln Pro Pro Thr Val Ala Gly Cys Ala Leu 2300 2305 2310Pro Pro Pro Lys Lys Ala Pro Thr Pro Pro Pro Arg Arg Arg Arg 2315 2320 2325Thr Val Gly Leu Ser Glu Ser Thr Ile Ser Glu Ala Leu Gln Gln 2330 2335 2340Leu Ala Ile Lys Thr Phe Gly Gln Pro Pro Ser Ser Gly Asp Ala 2345 2350 2355Gly Ser Ser Thr Gly Ala Gly Ala Ala Glu Ser Gly Gly Pro Thr 2360 2365 2370Ser Pro Gly Glu Pro Ala Pro Ser Glu Thr Gly Ser Ala Ser Ser 2375 2380 2385Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Glu Ser 2390 2395 2400Asp Gln Val Glu Leu Gln Pro Pro Pro Gln Gly Gly Gly Val Ala 2405 2410 2415Pro Gly Ser Gly Ser Gly Ser Trp Ser Thr Cys Ser Glu Glu Asp 2420 2425 2430Asp Thr Thr Val Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala 2435 2440 2445Leu Ile Thr Pro Cys Ser Pro Glu Glu Glu Lys Leu Pro Ile Asn 2450 2455 2460Pro Leu Ser Asn Ser Leu Leu Arg Tyr His Asn Lys Val Tyr Cys 2465 2470 2475Thr Thr Ser Lys Ser Ala Ser Gln Arg Ala Lys Lys Val Thr Phe 2480 2485 2490Asp Arg Thr Gln Val Leu Asp Ala His Tyr Asp Ser Val Leu Lys 2495 2500 2505Asp Ile Lys Leu Ala Ala Ser Lys Val Ser Ala Arg Leu Leu Thr 2510 2515 2520Leu Glu Glu Ala Cys Gln Leu Thr Pro Pro His Ser Ala Arg Ser 2525 2530 2535Lys Tyr Gly Phe Gly Ala Lys Glu Val Arg Ser Leu Ser Gly Arg 2540 2545 2550Ala Val Asn His Ile Lys Ser Val Trp Lys Asp Leu Leu Glu Asp 2555 2560 2565Pro Gln Thr Pro Ile Pro Thr Thr Ile Met Ala Lys Asn Glu Val 2570 2575 2580Phe Cys Val Asp Pro Ala Lys Gly Gly Lys Lys Pro Ala Arg Leu 2585 2590 2595Ile Val Tyr Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala 2600 2605 2610Leu Tyr Asp Ile Thr Gln Lys Leu Pro Gln Ala Val Met Gly Ala 2615 2620 2625Ser Tyr Gly Phe Gln Tyr Ser Pro Ala Gln Arg Val Glu Tyr Leu 2630 2635 2640Leu Lys Ala Trp Ala Glu Lys Lys Asp Pro Met Gly Phe Ser Tyr 2645 2650 2655Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Arg Asp Ile Arg 2660 2665 2670Thr Glu Glu Ser Ile Tyr Gln Ala Cys Ser Leu Pro Glu Glu Ala 2675 2680 2685Arg Thr Ala Ile His Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly 2690 2695 2700Pro Met Phe Asn Ser Lys Gly Gln Thr Cys Gly Tyr Arg Arg Cys 2705 2710 2715Arg Ala Ser Gly Val Leu Thr Thr Ser Met Gly Asn Thr Ile Thr 2720 2725 2730Cys Tyr Val Lys Ala Leu Ala Ala Cys Lys Ala Ala Gly Ile Val 2735 2740 2745Ala Pro Thr Met Leu Val Cys Gly Asp Asp Leu Val Val Ile Ser 2750 2755 2760Glu Ser Gln Gly Thr Glu Glu Asp Glu Arg Asn Leu Arg Ala Phe 2765 2770 2775Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro 2780 2785 2790Arg Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn 2795 2800 2805Val Ser Val Ala Leu Gly Pro Arg Gly Arg Arg Arg Tyr Tyr Leu 2810 2815 2820Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr 2825 2830 2835Val Arg His Ser Pro Ile Asn Ser Trp Leu Gly Asn Ile Ile Gln 2840 2845 2850Tyr Ala Pro Thr Ile Trp Val Arg Met Val Leu Met Thr His Phe 2855 2860 2865Phe Ser Ile Leu Met Val Gln Asp Thr Leu Asp Gln Asn Leu Asn 2870 2875 2880Phe Glu Met Tyr Gly Ser Val Tyr Ser Val Asn Pro Leu Asp Leu 2885 2890 2895Pro Ala Ile Ile Glu Arg Leu His Gly Leu Asp Ala Phe Ser Met 2900 2905 2910His Thr Tyr Ser His His Glu Leu Thr Arg Val Ala Ser Ala Leu 2915 2920 2925Arg Lys Leu Gly Ala Pro Pro Leu Arg Val Trp Lys Ser Arg Ala 2930 2935 2940Arg Ala Val Arg Ala Ser Leu Ile Ser Arg Gly Gly Lys Ala Ala 2945 2950 2955Val Cys Gly Arg Tyr Leu Phe Asn Trp Ala Val Lys Thr Lys Leu 2960 2965 2970Lys Leu Thr Pro Leu Pro Glu Ala Arg Leu Leu Asp Leu Ser Ser 2975 2980 2985Trp Phe Thr Val Gly Ala Gly Gly Gly Asp Ile Phe His Ser Val 2990 2995 3000Ser Arg Ala Arg Pro Arg Ser Leu Leu Phe Gly Leu Leu Leu Leu 3005 3010 3015Phe Val Gly Val Gly Leu Phe Leu Leu Pro Ala Arg 3020 3025 3030818DNAArtificialprimer -21M13 8tgtaaaacga cggccagt 18940DNAArtificialprimer MS98 9ggtttaggat tcgtgctcat ggtgcacggt ctacgagacc 401040DNAArtificialprimer MS97 10ggtctcgtag accgtgcacc atgagcacga atcctaaacc 401140DNAArtificialprimer MS96 11agatagcaca accaccacag gagcttggcg aggaatgcct 401240DNAArtificialprimer MS99 12agctgttcct cgctaggctc ctgtggtggt tgtgctatct 401320DNAArtificialprimer MS89 13cagctaccga ggggttaagc 201424DNAArtificialprimer MS151 14cccgggtacc cttggcccct ctat 241530DNAArtificialprimer MS165 15gtagggctgt tgtagctgac cagttcatca 301630DNAArtificialprimer MS164 16tgatgaactg gtcagctaca acagccctac 301722DNAArtificialprimer MS156 17tgggtggtac ccactcctga at 221830DNAArtificialprimer MS163 18gtagggctgt tgtagttgac cagttcatca 301930DNAArtificialprimer MS162 19tgatgaactg gtcaactaca acagccctac 30