Fusion protein comprising albumin and retinol-binding protein

SEQUENCE LISTING

The instant application contains a Sequence Listing which identical to the sequence listing of the parent application Ser. No. 15/001,720, filed Jan. 20, 2016.

TECHNICAL FIELD

The present invention relates to a fusion protein comprising albumin and retinol-binding protein, which is capable of being used for preventing or treating fibrotic diseases occurring in the liver, pancreas, lung, or other organs.

BACKGROUND ART

Tissue fibrosis leads to fatal defunctionalization of tissues. For example, liver fibrosis progresses to hepatocirrhosis, liver failure and liver cancer, and fibrosis in kidney ultimately leads to end-stage renal failure. Nevertheless, up to now, there have been no drugs for treating fibrotic diseases, and tissue grafting is the only cure available. The reason that there are no anti-fibrotic drugs is because molecular mechanism of fibrogenesis has not been clearly elucidated.

It is widely accepted that activated stellate cells play a key role in the development of fibrosis of liver and pancreas and that they are primarily responsible for the excessive deposition of extracellular matrix proteins such as collagen. It appears that, in addition to the liver, stellate cells are present in extrahepatic organs such as the pancreas, lung, kidney, intestine, spleen, salivary gland, and eye.

The stellate cells are important in controlling retinoid homeostasis in the whole body. Vitamin A (retinol), acquired from diet, is transferred to the liver and taken up by hepatocytes as a chylomicron remnant. It has been suggested that retinol binding protein (RBP) plays a role in the transfer of retinol from hepatocytes to hepatic stellate cells via a RBP receptor STRA6. Vitamin A is then stored as retinyl ester in cytoplasmic fat droplets in stellate cells. The present inventors disclosed that albumin is endogenously expressed in the stellate cells and involved in the formation of vitamin A-containing fat droplets, inhibiting stellate cell activation. The forced expression of albumin led to the phenotypic conversion from activated myofibroblast-like cells to quiescent fat-storing phenotype (Non-Patent Document 1: Kim N, Yoo W, Lee J, Kim H, Lee H, Kim Y, Kim D, Oh J.* (2009) Formation of vitamin A fat droplets in pancreatic stellate cells requires albumin. Gut 58(10), 1382-90; Non-Patent Document 2: Kim N, Choi S, Lim C, Lee H, Oh J. (2010) Albumin mediates PPAR-g and C/EBP-a-induced phenotypic changes in pancreatic stellate cells. Biochem. Biophys. Res. Commun. 391(1), 640-44.).

The interaction between tumor cells and their microenvironment has been recognized to affect cancer development by triggering cell proliferation and survival as well as the capability to invade the surrounding tissue (Thompson et al. Hepatic stellate cells: central modulators of hepatic carcinogenesis. BMC Gastroenterol. 2015 May 27; 15:63; Pothula et al. Key role of pancreatic stellate cells in pancreatic cancer. Cancer Lett. 2016 Oct. 10; 381(1):194-200). Studies in vitro and in vivo have provided evidence that activated stellate cells increase tumor cell migration, proliferation and produce a growth permissive environment that facilitates cancer progression (Vonlaufen et al. Pancreatic stellate cells: partners in crime with pancreatic cancer cells. Cancer Res. 2008 Apr. 1; 68(7):2085-93; Amann et al. Activated hepatic stellate cells promote tumorigenicity of hepatocellular carcinoma. Cancer Sci. 2009 April; 100(4):646-53; Okabe et al. Hepatic stellate cells accelerate the malignant behavior of cholangiocarcinoma cells. Ann Surg Oncol. 2011 April; 18(4):1175-84). We have performed experiments to show that the fusion protein R-III has the regulatory effects on tumor cell behavior through inhibiting the activation of stellate cells.

DISCLOSURE

Technical Problem

An object of the present invention is to treat fibrotic diseases via increasing the intracellular levels of full-length albumin or its partial protein (domain), which exerts the anti-fibrotic activity by inhibiting stellate cell activation or inducing aging of stellate cells.

Another object of the present invention is to treat a cancer via inhibiting stellate cells activation.

Technical Solution

In order to achieve the above object, an exemplary embodiment of the present invention provides a fusion protein comprising albumin and retinol-binding protein (RBP).

Albumin is a multifunctional plasma protein that is primarily synthesized by liver cells. It contributes to the maintenance of oncotic pressure as well as to transport of hydrophobic molecules. Albumin has three domains, each of which consists of two small sub-domains: A and B. According to a crystallographic analysis, five principal fatty acid binding sites are asymmetrically distributed within the albumin (one in sub-domain IB, one between IA and IIA, two in IIIA, and one in IIIB).

The present inventors hypothesized that albumin endogenously expressed in stellate cells may be involved in maintaining the quiescent vitamin A-storing phenotype by inhibiting stellate cell activation. On the other hand, the present inventors noticed that intravenously injected RBP protein is internalized into stellate cells via receptor-mediated endocytosis. In reference to this point, fusion protein comprising albumin (functional domain) and RBP (targeting domain) was developed and the therapeutic potential of fusion protein was then tested. As a result, when activated stellate cells were transfected with the expression vector encoding fusion protein, cells underwent phenotypic reversion to quiescent fat-storing cells, which was accompanied with the decreased in .alpha.-SMA levels, a maker for activated stellate cells. Furthermore, when conditioned medium of 293 cells transfected with the fusion protein expression vector was prepared and added to activated stellate cells, the fusion protein was found to be successfully incorporated into the stellate cells, induce the reappearance of cytoplasmic fat droplets, and reduce et-SMA levels. As previously reported with the mutant albumin, in which three high-affinity fatty acid binding sites (Arg410, Tyr411, and Lys525), located in domain III, were substituted with an alanine residue, the expression of the mutant fusion protein having triple point mutation also produces a senescence phenotype in stellate cells.

According to the present invention, the albumin sequence used for the construction of fusion protein may be derived from any species, but may be preferably derived from humans, in order to avoid a risk of immunogenicity. Albumin may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 1, but the present invention is not limited thereto.

Since intact high-affinity fatty acid binding sites in albumin protein are required for its anti-fibrotic activity and they are asymmetrically distributed in domain I and domain III, it may be preferable to use domain I and/or domain III as fusion protein component, but the present invention is not limited thereto. Therefore, according to a specific example, the albumin used for the construction of fusion protein may be at least one of an albumin I domain and albumin III domain.

The albumin I domain may be encoded by a nucleic acid sequence (1st to 666th nucleic acids among the nucleic acids encoding albumin) as set forth in SEQ ID NO: 2, but the present invention is not limited thereto.

In addition, the albumin III domain may have a nucleic acid sequence (1216th to 1827th nucleic acids among the nucleic acids encoding albumin) as set forth in SEQ ID NO: 3, but the present invention is not limited thereto.

According to a specific example, in the case of locating the albumin III at a N-terminal side of the fusion protein, an albumin N-terminal including a secretory sequence may be added before the albumin III domain. The albumin N-terminal may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 4, but the present invention is not limited thereto.

Meanwhile, for the RBP bound to the albumin, the full sequence of the RBP or a part of the full sequence of the RBP may be used, and may be properly selected according to an albumin sequence to be hound or an order of binding with the albumin. The RBP may be encoded by a nucleic acid sequence (1st to 585th nucleic acids among the nucleic acid encoding the RBP) as set forth in SEQ ID NO: 5, a nucleic acid sequence (55th to 585th nucleic acids among the nucleic acid encoding the RBP) as set forth in SEQ ID NO: 6, or a nucleic acid sequence (55th to 603th nucleic acids among the nucleic acid encoding the RBP) as set forth in SEQ ID NO: 7, but the present invention is not limited thereto. For example, since when the RBP is bound to the C-terminal of the albumin, a secretory sequence may not be needed, the RBP peptide encoded by a nucleic acid as set forth in SEQ ID NO: 6 or SEQ ID NO: 7 may be used. In addition, in a case where an albumin domain is again bound to the C-terminal of the RBP, it may be preferable to use a partial peptide of the RBP encoded by a nucleic acid sequence as set forth in SEQ ID NO: 7 rather than the full sequence of the RBP.

According to a preferable specific example, the fusion protein may be albumin I domain-RBP-albumin III, albumin III-RBP-albumin I, RBP-albumin albumin III-RBP, an albumin-RBP, or RBP-albumin. The albumin I domain-RBP-albumin III is a type in which the N-terminal of the RBP is bound to the C-terminal of albumin I domain and the albumin III domain is bound to the C-terminal of the RBP. The RBP-albumin III, albumin III-RBP, albumin-RBP, and RBP-albumin are also interpreted in the same way. From the above-mentioned sequence analysis, the present inventors found that the native conformations of albumin and RBP protein are conserved in the fusion protein of the above-mentioned type, and that fatty acid binding and RBP-RBP receptor binding are not affected. Therefore, the fusion protein may have one of the amino acid sequences set forth in SEQ ID NO: 8 to SEQ ID NO: 13, but the present invention is not limited thereto. Specifically, the albumin I domain-RBP-albumin Ill, albumin III-RBP-albumin I, RBP-albumin albumin III-RBP, albumin-RBP, and RBP-albumin may have amino acid sequences set forth in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively. These sequences may include peptide sequence for the proteins to be fused and also amino acid residues generated from the addition of endonuclease restriction site, His tag, and the like.

According to another specific example, wild-type albumin, or the wild-type albumin having partially substituted amino acids, may be used to induce aging of stellate cells by the variation of albumin. According to a specific example, for the albumin or albumin III domain included in the fusion protein, Arg410, Tyr411, and Lys525 may be substituted with Ala, but the present invention is not limited thereto.

In addition, the present invention provides a polynucleotide encoding the above-mentioned fusion protein comprising albumin and RBP, a recombinant vector including the polynucleotide, and a transformant containing the recombinant vector.

According to the present invention, the polynucleotide may have nucleic acid sequences set forth in SEQ ID NO: 14 to SEQ ID NO: 19, but the present invention is not limited thereto. Specifically, albumin I domain-RBP-albumin III, albumin III-RBP-albumin I, RBP-albumin III, albumin III-RBP, albumin-RBP, and RBP-albumin may be respectively encoded by nucleic acid sequences set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, but the present invention is not limited thereto. These sequences may include nucleic acids encoding the proteins to be fused, nucleic acids encoding restriction sites used for fusing proteins, a His tag for purifying, a stop codon, and the like.

According to another specific example, wild-type albumin, or the wild-type albumin having a partially substituted amino acid sequence among the amino acid sequences of the domain thereof may be used to induce aging of stellate cells by variation of the albumin. According to a specific example, for the albumin or albumin III domain included in the fusion protein, Arg410, Tyr411, and Lys525 may be substituted by Ala, but the present invention is not limited thereto.

Meanwhile, the recombinant vector including the polynucleotide may be prepared by inserting the polynucleotide into the known expression vector capable of being used for preparing a fusion protein. In the present invention, the term "vector" means a DNA construct including a DNA sequence operably bound to a proper regulatory sequence capable of expressing DNA in a proper host. A vector may be plasmid, phage particles, or simply a potential genome insert. In the case of transforming into a proper host, the vector can be replicated and can function regardless of a host genome, or may be integrated into the genome itself in some cases. Recently, plasmid is a type that is most generally used as a vector, so that in the present specification, "plasmid" and "vector" are used interchangeably. For purposes of the present invention, a plasmid vector is preferably used. A typical plasmid vector capable of being used for these purposes has (a) a replication origin that allows it to be effectively replicated to include hundreds of plasmid vectors per host cell, (b) antibiotic resistance genes allowing the host cell transformed into the plasmid vector to be selected, and (c) a structure including restriction enzyme cleavage sites capable of receiving insertion of an external DNA fragment. Even if there are no proper restriction enzyme cleavage sites, when a synthetic oligonucleotide adaptor or linker according to the general method is used, the vector and external DNA may be easily ligated.

Meanwhile, such a recombinant vector may include an expression vector allowing a His tag to be expressed at the end of a fusion protein in order to effectively isolate and purify a protein.

A host cell may be transformed by using a polynucleotide encoding a fusion protein including albumin and RBP and a recombinant vector including the polynucleotide. The host cell used for expressing a fusion protein according to the present invention may include a cancer cell, but the present invention is not limited thereto.

In addition, the present invention provides a method of producing a fusion protein in which comprises albumin and RBP comprising expressing the fusion protein in which comprises the albumin and RBP from the transformant. The expression of the fusion protein from the transformant may be generally induced through culturing the host cell. The fusion protein comprising the albumin and REP according to the present invention may be isolated from a culture medium by a known method for purifying a protein because the albumin and RBP themselves have a secretory signal, and thus the fusion protein is secreted out of the cell.

In the present invention, content in connection with genetic engineering technologies will be more clear by the content as disclosed in the document by Sambrook, et al. (Sambrook, et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, N.Y. (2001)) and the document by Frederick, et al. (Frederick M. Ausubel et al., Current protocols in molecular biology volume 1, 2, 3, John Wiley & Sons, Inc. (1994)).

In addition, the present invention provides a pharmaceutical composition for preventing or treating fibrotic diseases, in which the composition contains a fusion protein in which comprises albumin and RBP as an effective ingredient; a use of the fusion protein comprising the albumin and RBP for preparing a medicine for preventing or treating fibrotic diseases; and a method for treating fibrotic diseases, comprising administering to a subject in need thereof a therapeutically effective dose of the fusion protein comprising the albumin and RBP.

According to a specific example, the fibrotic disease occurs in the liver, pancreas, lung, kidney, intestine, spleen, salivary gland, or eye, but the present invention is not limited thereto. Examples of the fibrotic disease capable of being prevented or treated by the fusion protein according to the present invention include liver fibrosis, chronic hepatitis, cirrhosis, chemotherapy-associated steatohepatitis (CASH), lung fibrosis, renal fibrosis, renal failure, pancreatic fibrosis, chronic pancreatitis, retinal fibrosis/gliosis, or salivary gland fibrosis.

It has been widely accepted that tumorigenesis is determined not only by malignant cells but also by microenvironment. Targeting the crosstalk between tumors cells and their microenvironment may also represent a promising therapeutic strategy. In vitro and in vivo studies have convincingly demonstrated that there is a close bi-directional interaction between activated, pancreatic stellate cells and pancreatic cancer cells and between activated, hepatic stellate cells and liver cancer cells. This interaction reportedly increases proliferation and migration of cancer cells and facilitates distant metastasis (Cedric Coulouarn et al., Journal of hepatology, 2014, vol. 60, pp. 1306-1309; Minoti V Apte et al. Journal of Gastroenterology and Hepatology, 2012, vol. 27 Suppl. 2, pp. 69-74; Alexandra I Thompson et al. BMC Gastroenterology, 2015, vol. 15, pp. 63).

According to a specific example, the fusion protein comprising the albumin and RBP can regulate tumor cell behavior by inhibiting stellate cells activation, implicating that R-Ill can be used as an anti-cancer agent which modulates tumor microenvironment.

Therefore, the present invention provides a pharmaceutical composition for preventing or treating a cancer, in which the composition contains a fusion protein in which comprises albumin and RBP as an effective ingredient; a use of the fusion protein comprising the albumin and RBP for preparing a medicine for preventing or treating a cancer; and a method for treating a cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective dose of the fusion protein comprising the albumin and RBP.

The cancer includes liver cancer, breast cancer, pancreatic cancer, lung cancer, or kidney cancer, but the present invention is not limited thereto.

The pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition by further including at least one pharmaceutically acceptable carrier for administration in addition to an effective ingredient. Preferably, a liquid solution for an injection is suitable.

For the composition to be formulated in a liquid solution, the pharmaceutically acceptable carrier may include, to be suitable for sterilization and for a living body, saline solution, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol. In combination with at least one of these components, and if necessary, other general additives, such as antioxidant, a buffer solution, and bacteristat may be added. In addition, a form of dose to be injected, such as an aqueous solution, suspension, and an emulsion, may be formulated by further adding diluents, dispersing agents, surfactants, binding agents, and a lubricant. Furthermore, it may be preferably formulated according to a disease or components by using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton 5 PA, as a proper method in the art.

The pharmaceutical composition of the present invention may be administrated in a general way through a route such as intravenous injection, intra-arterial injection, intraperitoneal injection, intramuscular injection, and intrasternal injection.

An effective dose of an effective ingredient of the pharmaceutical composition according to the present invention means the amount required for effectively preventing or treating diseases. Accordingly, the effective dose may be controlled according to various factors such as a type of disease, disease severity, types and contents of the effective ingredient and other ingredients of the composition, a form of administration, an age, body weight, general health conditions, sex and a diet of a patient, an administration time, an administration route, composition secretion rate, a treatment period, and drugs taken concurrently. For example, in the case of the adult, the fusion protein of the present invention may be administrated in doses of 10 ng/kg to 10 g/kg when administrated once a day or several times a day, but the present invention is not limited thereto.

According to the present invention, subjects may be human, orangutan, chimpanzee, mouse, rat, dog, cow, chicken, pig, goat, and sheep, but they are preferably used.

Effects of the Invention

The fusion protein comprising albumin and retinol-binding protein (RBP) according to the present invention can be used for preventing or treating fibrotic diseases occurring in the liver, pancreas, lung, or other organs by inhibiting the activation of stellate cells or by inducing cellular senescence in stellate cells.

DESCRIPTION OF DRAWINGS

FIG. 1A is a schematic diagram illustrating production of the fusion protein of RBP-albumin.sup.406-608a.a (domain III) (hereinafter, also referred to as R-III) and albumin.sup.1-222 (domain I)-RBP-albumin.sup.406-608 (referred to as I-R-III), and FIG. 1B shows the result of western blot analysis assessing the effect of fusion protein expression on activated stellate cells.

FIGS. 2A to 2D show the effect of the expression of albumin or fusion protein on the morphology of activated stellate cells; phase contrast image (the left top panel), autofluorescence image (the right top panel), immunofluorescence (the left bottom panel), and oil red O staining (the right bottom panel).

FIG. 3 shows morphological changes in stellate cells transfected with the expression vector for the mutant fusion protein (R-III) having triple point mutation (R410A/Y411A/K525A).

FIG. 4A shows the result of western blot analysis illustrating that the fusion proteins according to the present invention are incorporated into stellate cells and induce biochemical changes, FIG. 4B shows the result of oil red O staining, describing the reappearance of cytoplasmic lipid droplets by fusion protein, and FIG. 4C shows the result of western blot analysis indicating that the fusion protein uptake is mediated through caveolae-mediated endocytosis.

FIG. 5A shows the purification process for the His-tagged, albumin-RBP fusion protein (R-III) according to the present invention using an ammonium sulfate precipitation (lane 1), a His Trap affinity column (lane 2), and a Resource Q column (lane 3), FIG. 5B shows the result of western blot analysis of hepatic tissue lysates after intravenous injection of R-III for 1 week, and FIG. 5C shows the result of in vivo experiment illustrating tissue distribution of R-III.

FIG. 6A shows the representative macroscopic pictures of livers from control and CCl.sub.4-, CCl.sub.4/albumin-, and CCl.sub.4/R-III-treated mice, and FIG. 6B shows the results of Sirius red staining and immunohistochemical analysis on liver sections for type I collagen (Scale bar, 200 .mu.m).

FIG. 7A shows the hydroxyproline content in the livers from control and CCl.sub.4-, CCl.sub.4/albumin-, and CCl.sub.4/R-III-treated mice (.mu.g/g liver) (*P=0.037, two-sample t-test (n=10) (CCl.sub.4+R-III compared to CCl.sub.4-treated mice)), FIG. 7B shows the results of western blot analysis of liver extracts prepared from the treated mice with use of anti-collagen type I antibody, and FIG. 7C shows the results of immunohistochemical analysis for .alpha.-SMA of liver sections from the treated mice (Scale bar, 200 .mu.m).

FIG. 8A shows the Sirius red staining results of liver sections from control and CCl.sub.4- and CCl.sub.4/R-III-treated mice (Scale bar, 200 .mu.m), and FIG. 8B shows the hydroxyproline content in the livers (.mu.g/g liver) (*P=0.034, two-sample t-test (n=10) (CCl.sub.4+R-III compared to CCl.sub.4-treated mice)).

FIG. 9A shows the Sirius red staining results of liver sections from control, bile duct ligation (BDL)-, and BDL/R-III-treated mice (Scale bar, 200 .mu.m), and FIG. 9B shows the hydroxyproline content in the livers (.mu.g/g liver) (*P-value, two-sample t-test (n=10) (compared to BDL-treated mice); BDL+R-III (10 .mu.g): 0.031, BDL+R-III (5 .mu.g): 0.026).

FIG. 10 shows the Masson's trichrome staining (FIG. 10A) and the results of immunohistochemical analysis for TGF-.beta.1 (FIG. 10B) and type I collagen (FIG. 10C) of kidney sections from control, UUO-, and UUO/R-III-treated mice.

FIG. 11 shows the results of immunohistochemical analysis for .alpha.-SMA (FIG. 11A) and desmin (FIG. 11B) of kidney sections from control, UUO-, and UUO/R-III-treated mice.

FIG. 12 shows the H&E staining (FIG. 12A) and the results of immunohistochemical analysis for type I collagen (FIG. 12B) of lung sections from control, bleomycin-, and bleomycin/R-III-treated mice, and FIG. 12C shows the hydroxyproline content in the lungs from control and bleomycin-, and bleomycin/R-III-treated mice (.mu.g/g lung).

FIG. 13 shows the results of immunohistochemical analysis for .alpha.-SMA (FIG. 13A) and TGF-.beta. (FIG. 13B) of lung sections from control, bleomycin-, and bleomycin/R-III-treated mice.

FIG. 14 shows effects of R-III on the mitogenic activity of conditioned media from activated hepatic stellate cells. One day after plating, mouse liver hepatoma Hepa-1c1c7 cells were incubated in CM from activated HSCs (.+-.R-III treatment), or control media (.+-.R-III). Cell proliferation was measured using the MIT assay. **P<0.01 vs. the control, #P<0.05 vs. CM from activated HSCs untreated with R-III.

FIG. 15 shows effects of R-III on the motility-stimulating activity of conditioned media from activated hepatic stellate cells. Hepa-1c1c7 cells were grown to confluence and a wound introduced in the monolayer using a pipette tip. After incubation in CM from activated HSCs (.+-.R-III treatment), or control media (.+-.R-III), relative migration distance of treated cell into the monolayer defect was measured. **P<0.01 vs. the control, #P<0.05 vs. CM from activated HSCs untreated with R-III.

BEST MODE

The above and other objects, features and advantages of the present invention will become clear by describing Examples below in detail. However, the present invention is not limited to the Examples described below, and can be implemented in various different forms. The following Examples are provided so that this disclosure will completely enable those of ordinary skill in the art to embody and practice the present invention.

EXAMPLE

<Experiment Method>

Isolation and Culture of Pancreatic Stellate Cells (PSCs)

Rat pancreatic stellate cells were isolated according to the method disclosed in Apte, M. V. et al., Periacinar stellate shaped cells in rat pancreas: identification, isolation, and culture. Gut 43 (1), 128-133 (1998). In summary, pancreas was finely minced, placed in a Hank's buffer solution containing 0.05% collagenase, 0.02% protease, and 0.1% DNase, and then shaken at 37.degree. C. for 20 minutes. After filtering through a 150 mm mesh, the cells were centrifuged by 13.2% Nycodenz gradient at 1400 g for 20 minutes. The pancreatic stellate cells were collected from the band just above the interface between the Nycodenz solution and aqueous layer, suspended in a DMEM (Dulbecco's modified Eagle's medium, Carlsbad, Calif.) supplemented with 10% fetal bovine serum, and then plated on a non-coated plastic dish. After reaching confluence in the primary culture, serial passages were obtained always applying 1:3 split.

Constitution of Expression Vector to Albumin-RBP Fusion Protein

Total RNA was extracted from a rat river tissue using a RNeasy kit (Qiagen, Valencia, Calif.) and reverse-transcribed into cDNA using GeneAmp RNA PCR (Applied Biosystems, Foster city, CA). The entire open reading frame (ORF) of albumin or RBP was amplified by polymerase chain reaction (PCR) with the designed primers and inserted into a pBluescript vector.

The expression vector encoding albumin I-RBP-albumin III (referred to as I-R-III) was prepared as follows. A DNA fragment encoding albumin (domain I: 1-666) (SEQ ID NO: 2) or a RBP (55-585) (SEQ ID NO: 6) was amplified from the pBluescript-albumin or pBluescript-RBP by PCR with the primers:

TABLE-US-00001 Albumin (domain I: 1-666) (SEQ ID NO: 2) Sense primer: (SEQ ID NO: 20) 5' GGGGTACCCC ACCATGAAGT GGGTAACCTT TC 3' Antisense primer: (SEQ ID NO: 21) 5' CCCCAATTGC ATCCTCTGAC GGACAGC 3' RBP (55-585) (SEQ ID NO: 6) Sense primer: (SEQ ID NO: 22) 5' GGGCAATTGG AGCGCGACTG CAGGGTG 3' Antisense primer: (SEQ ID NO: 23) 5' CCCCTCGAGT CTGCTTTGAC AGTAACC 3'.

The PCR products were double digested with KpnI/MFeI or MfeI/XhoI, respectively, and the DNA fragments purified by an agarose gel electrophoresis were ligated together and then cloned into KpnI/XhoI-cut pBluescript vector to yield pBluescript-I-R.

A DNA fragment encoding albumin (domain III: 1216-1827) (SEQ ID NO: 3) was amplified with the following primers:

TABLE-US-00002 Sense primer: (SEQ ID NO: 24) 5'GGGCTCGAGGAAGAACCTAAGAACTTG 3' Antisense primer: (SEQ ID NO: 25) 5' GGCTCTAGAT TAATGATGAT GATGATGATGGGCTAAGGCT TCTTTGCT 3'.

A His-tag sequence was included in the antisense primer. The PCR products were double digested with XhoI/XbaI and then ligated with the DNA fragment of I-R prepared above. The resulting DNA fragment I-R-III was inserted into expression vector pcDNA3.1+ at KpnI and XbaI sites to yield pcDNA3.1-I-R-III.

An expression vector encoding RBP-albumin III (referred to as R-III) was prepared as follows. A DNA fragment encoding RBP (1-585) (SEQ ID NO: 5) was amplified with the following primers.

TABLE-US-00003 Sense primer: (SEQ ID NO: 26) 5' GCGGAATTCC ACCATGGAGT GGGTGTGGGC 3' Antisense primer: (SEQ ID NO: 27) 5' CCCCTCGAGT CTGCTTTGAC AGTAACC 3'

The PCR products were double digested with EcoRI/XhoI, ligated with a DNA fragment encoding albumin (domain III: 1216-1827) (SEQ ID NO: 3), and then inserted into pcDNA3.1+ vector at EcoRI and XbaI sites to yield pcDNA3.1-R-III.

In the pcDNA3.1-I-R-III or pcDNA3.1-R-III, an albumin/RBP encoding region was located immediately upstream of 6-histidine tag encoding sequence and stop codon in the same reading frame.

Meanwhile, it was reported that the expression of mutant albumin, in which three high-affinity fatty acid binding sites (Arg410, Tyr411, and Lys525) are substituted with an Ala residue, produces a senescence phenotype in stellate cells (Kim N, Yoo W, Lee J, Kim H, Lee H, Kim Y, Kim D, Oh J.* (2009) Formation of vitamin A fat droplets in pancreatic stellate cells requires albumin. Gut 58(10), 1382-90.). It indicates that direct interaction with lipophilic substances is important for the action of albumin in stellate cells. Accordingly, in the present invention, the expression vector for mutant fusion protein having triple point mutation (R410A/Y411A/K525A) was prepared by a PCR-based method using Muta-Direct.TM. Site-Directed Mutagenesis Kit (iNtRON, Korea); the expression vector was transfected into the activated stellate cells; and then phenotypic changes were examined.

All the constructs were sequenced by using an auto-sequencer to confirm an albumin/RBP encoding region.

Purification of (his) 6 Tagged Recombinant Fusion Protein

An expression vector encoding mouse R-III was prepared in the same manner as the rat fusion protein. Primers used for a PCR were as follows.

TABLE-US-00004 Albumin (domain III: 1216-1827) (SEQ ID NO: 3) Sense primer: (SEQ ID NO: 28) 5' GGGCTCGAGG AAGAGCCTAA GAACTTG 3' Antisense primer: (SEQ ID NO: 29) 5' GGCTCTAGAT TAATGATGAT GATGATGATGGGCTAAGGTG TCTTTGCA 3' RBP (1-585) (SEQ ID NO: 5) Sense primer: (SEQ ID NO: 30) 5' GCGGAATTCC ACCATGGAGT GGGTGTGGGC 3' Antisense primer: (SEQ ID NO: 31) 5' CCCCTCGAGC CTGCTTTGAC AGTAACC 3'

293 cells were stably transfected with an expression vector encoding mouse R-III, and the high expressing, clonal cell lines were selected by assessing levels of secreted R-III by western blotting using an anti-His tag antibody. Culture medium of 293 cells was fractionated with ammonium sulfate (55%), then subject to His Trap affinity column. The sample was further purified by a Resource Q. The purified proteins were dialyzed with deionized water, freeze-dried, and then dissolved in saline solution. As determined by SDS-PAGE and protein staining, the purity of R-III was above 95%.

Transfection

Activated pancreas stellate cells (after passage 2) was transiently transfected using lipofectamine 2000 (Invitrogen, Carlsbad, Calif.), and then after 24 hours, the cells were analyzed.

Western Blotting Analysis

The cells were rinsed in ice-cold phosphate buffer saline (PBS) twice, and harvested by scraping in a lysis buffer solution. The equivalent amounts of proteins were separated by a SDS-PAGE, followed by immunoblot detection using a primary antibody. The primary antibodies were as follows: albumin (Santa Cruz, Santa Cruz, Calif.), .alpha.-SMA (Sigma, St. Louis, Mo.), .alpha.-tubulin (Cell signaling, Beverly, Mass.) and Type I collagen (Calbiochem, San Diego, Calif.), His-tag (AB Frontier, Seoul, Korea).

Immunofluorescence Analysis

Pancreatic stellate cells were plated on a glass cover slip coated with gelatin. The samples were fixed with paraformaldehyde, incubated with an albumin antibody (Santa Cruz #sc-58698) overnight at 4.degree. C. in a moist chamber, and reacted with a secondary antibody bound with Alexa Fluor 568. The cells were washed with PBS and mounted onto a slide. The stained cells were visualized by using Zeiss AXIO Imager M1 microscope.

Oil Red O Staining

Fat droplets were visualized by staining the pancreatic stellate cells with oil red O using the method disclosed by Koopman (Koopman, R., Schaart, G., & Hesselink, M. K., Optimisation of oil red O staining permits combination with immunofluorescence and automated quantification of lipids. Histochem Cell Biol 116 (1), 63-68 (2001)). The oil red O was diluted in triethyl phosphate instead of isopropane.

Preparation of Liver Fibrosis Model

For CCl.sub.4-induced liver fibrosis study, BALB/c mice were treated with CCl.sub.4 (1 ml/kg body weight; 1:1 dilution with mineral oil) or mineral oil as a control by intraperitoneal (i.p.) injection three times per week for 7 weeks. For the determination of therapeutic effects of albumin-RBP fusion protein R-III, CCl.sub.4-treated mice were randomly divided into three groups; mice were administered via tail vein injection with saline, albumin (10 .mu.g) or R-III (10 .mu.g) every day during the last 2 weeks of CCl.sub.4 treatment. For the assessment of preventive effects of CCl.sub.4-treated mice were randomly divided into two groups and administered with saline or R-III three times per week over a period of 7 weeks. Three to five mineral/saline-treated mice were used as normal controls for each experiment. For the study of liver fibrosis induced by bile duct ligation (BDL), mice were anesthetized intraperitoneally by ketamine and xylazine. After midline laparotomy, the common bile duct was double-ligated and transected between the ligatures. The sham operation was performed similarly without BDL. R-III (0, 1, 5, or 10 .mu.g) was daily administered, beginning on day 7 after BDL, for 2 weeks. All experiments were repeated twice.

Preparation of Kidney Fibrosis Model

UUO (unilateral ureteral obstruction) model was performed by using a BALB/c mouse. In summary, the abdominal cavity of the mouse was opened through a midline incision, and then the left ureter was isolated and tied up. By a similar method, sham-operated animals were subjected to the same surgical operation, but the ureter ligation was not performed. In order to test a therapeutic effect of R-III on improving kidney fibrosis, R-III (10 .mu.g) was daily administrated, beginning on day 6 after blocking, for 7 days through tail vein injection. All mice were sacrificed under anesthesia at day 14 and kidney tissues were weighed and collected for various analysis. The half of the kidney was fixed with 10% buffer formalin in order for a histological study, and the other half was snap-frozen in liquid nitrogen to store at -80.degree. C. in order for extractions of protein and RNA. During the experiment, food intake, water intake, urine volume, body weight levels were measured at the baseline and at the time of sacrifice.

Preparation of Lung Fibrosis Model

For bleomycin-induced pulmonary fibrosis study, BALB/c mice were anesthetized with inhalational isoflurane using isoflurane vaporizer. Bleomycin sulfate was dissolved in filtered phosphate-buffered saline (PBS) solution and applied by intratracheal instillation as a single dose of 0.08 units per mouse. Bleomycin-treated mice were randomly divided into two groups; mice were administered via tail vein injection with saline or R-III (10 .mu.g) daily, beginning on day 7 after bleomycin, for 10 days. Mice were then sacrificed on day 18. Lungs were excised and stored at -80.degree. C. for biochemical assays and in 10% neutral buffered formalin for histological analysis.

Immunohistological Analysis

The section (5 .mu.m thickness) of formalin-fixed, paraffin-embedded liver tissues were prepared, stained with a H&E for a histological analysis and with Sirius red or Masson's trichrome for collagen deposition. In addition, the tissue sections were immunohistochemically stained with Type I collagen (Abeam, Cambridge, UK) antibody. In order to quantify the Sirius red staining, Image J software (NIH) was used.

Collection of conditioned medium (CM) from activated mouse hepatic stellate cells (HSCs)

One day after plating into T75 flasks, activated mouse HSCs were washed with serum-free DMEM, and then incubated for 24 h with serum-free DMEM (10 mL/T75) in the presence or absence of R-III (150 nM). Scrum-free DMEM (with/without R-III) incubated for 24 h in cell culture flasks without cells served as the controls.

Proliferation Assay

Mouse liver hepatoma Hepa-1c1c7 cells were seeded into 96-well plates (4000 cells/well) in DMEM supplemented with 10% FBS. One day later, cells were washed with serum-free DMEM and subsequently cultured in CM from activated HSC or control media. Cell proliferation was measured using the MTT assay. Experiments were carried out in triplicate and were repeated three times.

Migration Assays

Migration of Hepa-1c1c7 cells was assessed by wound-healing-assay. Briefly, cells were plated in high density into 12-well plates. After adherence, cells were incubated either in CM from activated HSC or control media. A wound was introduced by scratching the confluent monolayer with a pipette tip, and the migration was measured after 24 and 48 h. Each analysis was performed in triplicate and repeated twice.

Statistical Analysis

The results were expressed as mean.+-.standard deviation (SD). A statistical analysis was performed by using t-tests. Comparisons were considered significant at P<0.05, and the P values were two-tailed.

<Experimental Result>

Induction of lipid droplet formation in pancreatic stellate cells by albumin-RBP fusion protein

In the aforementioned RBP-albumin.sup.406-608 a.a (domain III) (R-III) and albumin.sup.1-222 (domain I)-RBP-albumin.sup.406-608 (I-R-III) (FIG. 1A), polyhistidine tag was placed on C-terminal of the fusion proteins to facilitate purification. After pancreatic stellate cells were activated after passage 2, cells were transiently transfected with expression vector for wild-type albumin, R-III or I-R-III and the effects were then examined. Western blotting revealed that fusion proteins have expected size (R-III.about.45 kDa and I-R-III.about.68 kDa) and decrease the levels of .alpha.-SMA and collagen type 1, markers of activated stellate cells (FIG. 1B).

FIGS. 2A to 2D show the morphological changes in stellate cells by the expression of fusion protein; phase contrast image (left top panel), autofluorescence image (right top panel), immunofluorescence (left bottom panel), and oil red O staining (right bottom panel). Activated pancreatic stellate cells under normal culture conditions show a fibroblastoid morphology (FIG. 2A). Expression of wild-type albumin, R-III or I-R-III, however, led to the formation of autofluorescent fat droplets and induced phenotypic changes (FIGS. 2B to 2D), which is consistent with the above-mentioned Western blot data (FIG. 1B). This result suggests that the partial protein (domain III) can still induce stellate cell inactivation similar to the full-length albumin.

Meanwhile, as a result of experimenting with a mutant fusion protein having triple point mutation (R410A/Y411A/K525A) performed by the above-mentioned method, the expression of the mutant fusion protein produces a senescence phenotype, as manifested by enlarged cell size and flattened cell body (FIG. 3).

Cellular Uptake of Albumin-RBP Fusion Protein into Stellate Cells

In order to investigate whether the RBP moiety can enable cellular uptake of the fusion protein, conditioned medium was prepared from the 293 cells stably transfected with albumin, R-III, or I-R-III expression vector and applied to activated pancreatic stellate cells. Western blotting with use of anti-His tag antibody revealed that fusion proteins, but not full-length albumin, are successfully incorporated into stellate cells and reduce the levels of .alpha.-SMA and collagen type I (FIG. 4A). In addition, fusion proteins induced the reappearance of lipid droplets (FIG. 4B). We further investigated the mechanism of R-III internalization using inhibitor of clathrin-mediated endocytosis (chlorpromazine) or cavelolae-mediated endocytosis (filipin). Western blotting analysis revealed that cellular uptake of R-III was largely inhibited by filipin pre-treatment, which is consistent with the previous report that RBP enters the cell through caveolae-mediated endocytosis (FIG. 4C). Therefore, the data shows that each component of the fusion protein is functionally important. In other words, in the fusion protein, the RBP performs a role in stellate cell-targeting moiety and albumin domain performs a role in stellate cell-inactivating domain.

Tissue Distribution of Injected Albumin-RBP Fusion Protein

Tissue distribution of albumin-RBP fusion protein was investigated in vivo. Since R-III was abundantly expressed and secreted from the transfected 293 cells as compared with I-R-III (data not shown), R-III was selected and purified using FPLC to >95% purity (FIG. 5A). The R-III (3 or 10 .mu.g) dissolved in 0.1 ml of saline solution was injected every day into the tail vein of a BALB/c mouse for 7 days, and then liver lysate was analyzed by western blotting using anti-His tag antibody. The distinct R-III protein band was observed in the R-III-injected mouse, and the band intensity thereof increased in dose dependent manner (FIG. 5B). When equivalent amounts of whole cell lysates obtained from different tissues was analyzed by western blotting, strong R-III signal was observed in liver, and also a weak signal was detected in the brain, lung, spleen, pancreas, kidney, and intestine (FIG. 5C). Such tissue distribution of R-III appears to be similar to that of RBP.

Effect of R-III on Inhibiting Liver Fibrosis

On the basis of the in vitro anti-fibrotic activity, we explored the therapeutic effects of R-III on CCl.sub.4-induced liver fibrosis model. The external surface of the liver in mineral oil/saline-treated control mice was smooth and glistening, while multiple nodules were found macroscopically on the surfaces of livers in CCl.sub.4/saline-treated mice (FIG. 6A). Interestingly, R-III treatment significantly reduced nodule incidence, which was not observed in mice treated with albumin. The histological analysis of livers in the control mice showed normal architecture, whereas liver fibrosis was severe in CCl.sub.4/saline-treated mice, as evidenced by disruption of tissue architecture and large fibrous septa formation (FIG. 6B). Sirius red staining and immunohistochemistry also confirmed extensive collagen deposition in the liver (FIG. 6B). R-III significantly reduced histopathological alterations and collagen deposition. Hydroxyproline assays showed 35% reduction in collagen content by R-III, which was also confirmed by Western blot (FIGS. 7A and 7B). Intense immunostaining for .alpha.-smooth muscle actin (SMA) was found along the fibrotic septa around the central vein in CCl.sub.4/saline-treated livers, and R-III treatment considerably decreased .alpha.-SMA staining (FIG. 7C). To examine whether R-III has a preventive effect on CCl.sub.4-induced liver fibrosis, mice were treated with CCl.sub.4 and R-III on different days three times per week over a period of 7 weeks. Sirius red staining of liver sections showed that R-III treatment markedly reduced collagen deposition (FIG. 8A). Collagen content was reduced by 45% in the R-III-treated group, as measured using hydroxyproline assays (FIG. 8B). Mice underwent bile duct ligation (BDL) and were daily administered with R-III (1, 5 or 10 .mu.g) from 2 to 3 weeks of BDL. R-III treatment reduced cholestatic liver fibrosis (FIG. 9A) and reduced collagen content by up to 45% (FIG. 9B).

Kidney Fibrosis Decrease by R-III

We evaluated the therapeutic effects of R-III against unilateral ureteral obstruction (UUO)-induced renal fibrosis. Sham-operated control mice showed normal renal architecture (FIG. 10). UUO kidneys revealed increased interstitial fibrosis and tubular atrophy, while R-III treatment attenuated the degree of interstitial fibrosis, as evidenced by Masson's trichrome staining (FIG. 10A). Immunoreactivity with pro-fibrotic molecules such as TGF-.beta.1 and collagen type1 was also significantly decreased with R-III treatment (FIGS. 10B and 10C). Immunostaining for .alpha.-SMA and desmin, markers of myofibroblast formation, were diminished after R-III treatment compared with those in UUO kidneys (FIGS. 11A and 11B).

Lung Fibrosis Decrease by R-III

Intratracheal beomycin treatment significantly increased the alveolar septum infiltrates, inflammatory cell infiltrates, and collagen fibers as compared with control group (FIGS. 12A-C). These bleomycin-induced changes were significantly attenuated by R-III treatment. In addition, bleomycin significantly increased levels of lung fibrosis markers .alpha.-SMA and TGF-.beta. (FIGS. 13A and 13B). R-III treatment largely reversed bleomycin-induced changes in these lung fibrosis markers. Our results suggest that R-III may have potential therapeutic value for lung fibrosis treatment.

Effects of R-III on Hepa-1c17 Cell Proliferation

Analysis of cell proliferation showed that CM from activated mouse hepatic stellate cells (HSCs) enhanced proliferation of mouse liver hepatoma Hepa-1c1c7 cells (FIG. 14), while CM from R-III-treated HSCs had significantly less mitogenic activity. R-III itself had no effects on the proliferation of Hepa-1c1c7 cells.

Effects of R-III on Hepa-1c1c7 Cell Migration

Wound healing assay revealed that CM from activated HSCs promoted migration of Hepa-1c1c7 cells as compared with the control CM (FIG. 15). CM from R-III-treated HSCs is much less effective in promoting cell migration. R-III itself had no effects on the migration of Hepa-1c1c7 cells.

These findings show that R-III can regulate tumor cell behavior by inhibiting stellate cells activation, implicating that R-III can be used as an anti-cancer agent which modulates tumor microenvironment.

SEQUENCE LISTINGS

1

3511827DNAHomo sapiens 1atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60gtgtttcgtc gagatgcaca caagagtgag gttgctcatc ggtttaaaga tttgggagaa 120gaaaatttca aagccttggt gttgattgcc tttgctcagt atcttcagca gtgtccattt 180gaagatcatg taaaattagt gaatgaagta actgaatttg caaaaacatg tgttgctgat 240gagtcagctg aaaattgtga caaatcactt catacccttt ttggagacaa attatgcaca 300gttgcaactc ttcgtgaaac ctatggtgaa atggctgact gctgtgcaaa acaagaacct 360gagagaaatg aatgcttctt gcaacacaaa gatgacaacc caaacctccc ccgattggtg 420agaccagagg ttgatgtgat gtgcactgct tttcatgaca atgaagagac atttttgaaa 480aaatacttat atgaaattgc cagaagacat ccttactttt atgccccgga actccttttc 540tttgctaaaa ggtataaagc tgcttttaca gaatgttgcc aagctgctga taaagctgcc 600tgcctgttgc caaagctcga tgaacttcgg gatgaaggga aggcttcgtc tgccaaacag 660agactcaagt gtgccagtct ccaaaaattt ggagaaagag ctttcaaagc atgggcagta 720gctcgcctga gccagagatt tcccaaagct gagtttgcag aagtttccaa gttagtgaca 780gatcttacca aagtccacac ggaatgctgc catggagatc tgcttgaatg tgctgatgac 840agggcggacc ttgccaagta tatctgtgaa aatcaagatt cgatctccag taaactgaag 900gaatgctgtg aaaaacctct gttggaaaaa tcccactgca ttgccgaagt ggaaaatgat 960gagatgcctg ctgacttgcc ttcattagct gctgattttg ttgaaagtaa ggatgtttgc 1020aaaaactatg ctgaggcaaa ggatgtcttc ctgggcatgt ttttgtatga atatgcaaga 1080aggcatcctg attactctgt cgtgctgctg ctgagacttg ccaagacata tgaaaccact 1140ctagagaagt gctgtgccgc tgcagatcct catgaatgct atgccaaagt gttcgatgaa 1200tttaaacctc ttgtggaaga gcctcagaat ttaatcaaac aaaattgtga gctttttgag 1260cagcttggag agtacaaatt ccagaatgcg ctattagttc gttacaccaa gaaagtaccc 1320caagtgtcaa ctccaactct tgtagaggtc tcaagaaacc taggaaaagt gggcagcaaa 1380tgttgtaaac atcctgaagc aaaaagaatg ccctgtgcag aagactatct atccgtggtc 1440ctgaaccagt tatgtgtgtt gcatgagaaa acgccagtaa gtgacagagt caccaaatgc 1500tgcacagaat ccttggtgaa caggcgacca tgcttttcag ctctggaagt cgatgaaaca 1560tacgttccca aagagtttaa tgctgaaaca ttcaccttcc atgcagatat atgcacactt 1620tctgagaagg agagacaaat caagaaacaa actgcacttg ttgagctcgt gaaacacaag 1680cccaaggcaa caaaagagca actgaaagct gttatggatg atttcgcagc ttttgtagag 1740aagtgctgca aggctgacga taaggagacc tgctttgccg aggagggtaa aaaacttgtt 1800gctgcaagtc aagctgcctt aggctta 18272666DNAHomo sapiens 2atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60gtgtttcgtc gagatgcaca caagagtgag gttgctcatc ggtttaaaga tttgggagaa 120gaaaatttca aagccttggt gttgattgcc tttgctcagt atcttcagca gtgtccattt 180gaagatcatg taaaattagt gaatgaagta actgaatttg caaaaacatg tgttgctgat 240gagtcagctg aaaattgtga caaatcactt catacccttt ttggagacaa attatgcaca 300gttgcaactc ttcgtgaaac ctatggtgaa atggctgact gctgtgcaaa acaagaacct 360gagagaaatg aatgcttctt gcaacacaaa gatgacaacc caaacctccc ccgattggtg 420agaccagagg ttgatgtgat gtgcactgct tttcatgaca atgaagagac atttttgaaa 480aaatacttat atgaaattgc cagaagacat ccttactttt atgccccgga actccttttc 540tttgctaaaa ggtataaagc tgcttttaca gaatgttgcc aagctgctga taaagctgcc 600tgcctgttgc caaagctcga tgaacttcgg gatgaaggga aggcttcgtc tgccaaacag 660agactc 6663612DNAHomo sapiens 3gaagagcctc agaatttaat caaacaaaat tgtgagcttt ttgagcagct tggagagtac 60aaattccaga atgcgctatt agttcgttac accaagaaag taccccaagt gtcaactcca 120actcttgtag aggtctcaag aaacctagga aaagtgggca gcaaatgttg taaacatcct 180gaagcaaaaa gaatgccctg tgcagaagac tatctatccg tggtcctgaa ccagttatgt 240gtgttgcatg agaaaacgcc agtaagtgac agagtcacca aatgctgcac agaatccttg 300gtgaacaggc gaccatgctt ttcagctctg gaagtcgatg aaacatacgt tcccaaagag 360tttaatgctg aaacattcac cttccatgca gatatatgca cactttctga gaaggagaga 420caaatcaaga aacaaactgc acttgttgag ctcgtgaaac acaagcccaa ggcaacaaaa 480gagcaactga aagctgttat ggatgatttc gcagcttttg tagagaagtg ctgcaaggct 540gacgataagg agacctgctt tgccgaggag ggtaaaaaac ttgttgctgc aagtcaagct 600gccttaggct ta 612484DNAHomo sapiens 4atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60gtgtttcgtc gagatgcaca caag 845585DNAHomo sapiens 5atgaagtggg tgtgggcgct cttgctgttg gcggcgctgg gcagcggccg cgcggagcgc 60gactgccgag tgagcagctt ccgagtcaag gagaacttcg acaaggctcg cttctctggg 120acctggtacg ccatggccaa gaaggacccc gagggcctct ttctgcagga caacatcgtc 180gcggagttct ccgtggacga gaccggccag atgagcgcca cagccaaggg ccgagtccgt 240cttttgaata actgggacgt gtgcgcagac atggtgggca ccttcacaga caccgaggac 300cctgccaagt tcaagatgaa gtactggggc gtagcctcct ttctccagaa aggaaatgat 360gaccactgga tcgtcgacac agactacgac acgtatgccg tgcagtactc ctgccgcctc 420ctgaacctcg atggcacctg tgctgacagc tactccttcg tgttttcccg ggaccccaac 480ggcctgcccc cagaagcgca gaagattgta aggcagcggc aggaggagct gtgcctggcc 540aggcagtaca ggctgatcgt ccacaacggt tactgcgatg gcaga 5856531DNAHomo sapiens 6gagcgcgact gccgagtgag cagcttccga gtcaaggaga acttcgacaa ggctcgcttc 60tctgggacct ggtacgccat ggccaagaag gaccccgagg gcctctttct gcaggacaac 120atcgtcgcgg agttctccgt ggacgagacc ggccagatga gcgccacagc caagggccga 180gtccgtcttt tgaataactg ggacgtgtgc gcagacatgg tgggcacctt cacagacacc 240gaggaccctg ccaagttcaa gatgaagtac tggggcgtag cctcctttct ccagaaagga 300aatgatgacc actggatcgt cgacacagac tacgacacgt atgccgtgca gtactcctgc 360cgcctcctga acctcgatgg cacctgtgct gacagctact ccttcgtgtt ttcccgggac 420cccaacggcc tgcccccaga agcgcagaag attgtaaggc agcggcagga ggagctgtgc 480ctggccaggc agtacaggct gatcgtccac aacggttact gcgatggcag a 5317549DNAHomo sapiens 7gagcgcgact gccgagtgag cagcttccga gtcaaggaga acttcgacaa ggctcgcttc 60tctgggacct ggtacgccat ggccaagaag gaccccgagg gcctctttct gcaggacaac 120atcgtcgcgg agttctccgt ggacgagacc ggccagatga gcgccacagc caagggccga 180gtccgtcttt tgaataactg ggacgtgtgc gcagacatgg tgggcacctt cacagacacc 240gaggaccctg ccaagttcaa gatgaagtac tggggcgtag cctcctttct ccagaaagga 300aatgatgacc actggatcgt cgacacagac tacgacacgt atgccgtgca gtactcctgc 360cgcctcctga acctcgatgg cacctgtgct gacagctact ccttcgtgtt ttcccgggac 420cccaacggcc tgcccccaga agcgcagaag attgtaaggc agcggcagga ggagctgtgc 480ctggccaggc agtacaggct gatcgtccac aacggttact gcgatggcag atcagaaaga 540aaccttttg 5498613PRTHomo sapiens 8Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Gln Leu 210 215 220 Glu Arg Asp Cys Arg Val Ser Ser Phe Arg Val Lys Glu Asn Phe Asp 225 230 235 240 Lys Ala Arg Phe Ser Gly Thr Trp Tyr Ala Met Ala Lys Lys Asp Pro 245 250 255 Glu Gly Leu Phe Leu Gln Asp Asn Ile Val Ala Glu Phe Ser Val Asp 260 265 270 Glu Thr Gly Gln Met Ser Ala Thr Ala Lys Gly Arg Val Arg Leu Leu 275 280 285 Asn Asn Trp Asp Val Cys Ala Asp Met Val Gly Thr Phe Thr Asp Thr 290 295 300 Glu Asp Pro Ala Lys Phe Lys Met Lys Tyr Trp Gly Val Ala Ser Phe 305 310 315 320 Leu Gln Lys Gly Asn Asp Asp His Trp Ile Val Asp Thr Asp Tyr Asp 325 330 335 Thr Tyr Ala Val Gln Tyr Ser Cys Arg Leu Leu Asn Leu Asp Gly Thr 340 345 350 Cys Ala Asp Ser Tyr Ser Phe Val Phe Ser Arg Asp Pro Asn Gly Leu 355 360 365 Pro Pro Glu Ala Gln Lys Ile Val Arg Gln Arg Gln Glu Glu Leu Cys 370 375 380 Leu Ala Arg Gln Tyr Arg Leu Ile Val His Asn Gly Tyr Cys Asp Gly 385 390 395 400 Arg Leu Glu Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu 405 410 415 Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg 420 425 430 Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val 435 440 445 Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu 450 455 460 Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn 465 470 475 480 Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr 485 490 495 Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala 500 505 510 Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr 515 520 525 Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln 530 535 540 Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys 545 550 555 560 Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe 565 570 575 Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu 580 585 590 Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu His 595 600 605 His His His His His 610 9619PRTHomo sapiens 9Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Gln Leu Glu Glu 20 25 30 Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly 35 40 45 Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val 50 55 60 Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly 65 70 75 80 Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro 85 90 95 Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu 100 105 110 His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu 115 120 125 Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu 130 135 140 Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala 145 150 155 160 Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr 165 170 175 Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln 180 185 190 Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys 195 200 205 Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu 210 215 220 Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Leu Glu Glu Arg Asp Cys 225 230 235 240 Arg Val Ser Ser Phe Arg Val Lys Glu Asn Phe Asp Lys Ala Arg Phe 245 250 255 Ser Gly Thr Trp Tyr Ala Met Ala Lys Lys Asp Pro Glu Gly Leu Phe 260 265 270 Leu Gln Asp Asn Ile Val Ala Glu Phe Ser Val Asp Glu Thr Gly Gln 275 280 285 Met Ser Ala Thr Ala Lys Gly Arg Val Arg Leu Leu Asn Asn Trp Asp 290 295 300 Val Cys Ala Asp Met Val Gly Thr Phe Thr Asp Thr Glu Asp Pro Ala 305 310 315 320 Lys Phe Lys Met Lys Tyr Trp Gly Val Ala Ser Phe Leu Gln Lys Gly 325 330 335 Asn Asp Asp His Trp Ile Val Asp Thr Asp Tyr Asp Thr Tyr Ala Val 340 345 350 Gln Tyr Ser Cys Arg Leu Leu Asn Leu Asp Gly Thr Cys Ala Asp Ser 355 360 365 Tyr Ser Phe Val Phe Ser Arg Asp Pro Asn Gly Leu Pro Pro Glu Ala 370 375 380 Gln Lys Ile Val Arg Gln Arg Gln Glu Glu Leu Cys Leu Ala Arg Gln 385 390 395 400 Tyr Arg Leu Ile Val His Asn Gly Tyr Cys Asp Gly Arg Ser Arg Asp 405 410 415 Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu 420 425 430 Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln 435 440 445 Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe 450 455 460 Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser 465 470 475 480 Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg 485 490 495 Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu 500 505 510 Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro 515 520 525 Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp 530 535 540 Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg 545 550 555 560 His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr 565 570 575 Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys 580 585 590 Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser 595 600 605 Ala Lys Gln Arg Leu His His His His His His 610 615 10407PRTHomo sapiens 10Met Lys Trp Val Trp Ala Leu Leu Leu Leu Ala Ala Leu Gly Ser Gly 1 5 10 15 Arg Ala Glu Arg Asp Cys Arg Val Ser Ser Phe Arg Val Lys Glu Asn 20 25 30 Phe Asp Lys Ala Arg Phe Ser Gly Thr Trp Tyr Ala Met Ala Lys Lys 35 40 45 Asp Pro Glu Gly Leu Phe Leu Gln Asp Asn Ile Val Ala Glu Phe Ser 50 55 60 Val Asp Glu Thr Gly Gln Met Ser Ala Thr Ala Lys Gly Arg Val Arg 65 70 75 80 Leu Leu Asn Asn Trp Asp Val Cys Ala Asp Met Val Gly Thr Phe Thr 85 90 95 Asp Thr Glu Asp Pro Ala Lys Phe Lys Met Lys Tyr Trp Gly Val Ala 100 105 110 Ser Phe Leu Gln Lys Gly Asn Asp Asp His Trp Ile Val Asp Thr Asp 115 120 125 Tyr Asp Thr Tyr Ala Val Gln Tyr Ser Cys Arg Leu Leu Asn Leu Asp 130 135 140 Gly Thr Cys Ala Asp Ser Tyr Ser Phe Val Phe Ser Arg Asp Pro Asn 145 150 155 160 Gly Leu Pro Pro Glu Ala Gln Lys Ile Val Arg Gln Arg Gln Glu Glu 165 170 175 Leu Cys Leu Ala Arg Gln Tyr Arg Leu Ile Val His Asn Gly Tyr Cys 180 185 190 Asp Gly Arg Leu Glu Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 195 200 205 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 210 215 220 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 225 230 235 240 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 245 250 255 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val

260 265 270 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 275 280 285 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 290 295 300 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 305 310 315 320 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 325 330 335 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 340 345 350 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 355 360 365 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 370 375 380 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 385 390 395 400 Leu His His His His His His 405 11425PRTHomo sapiens 11Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Gln Leu Glu Glu 20 25 30 Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly 35 40 45 Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val 50 55 60 Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly 65 70 75 80 Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro 85 90 95 Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu 100 105 110 His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu 115 120 125 Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu 130 135 140 Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala 145 150 155 160 Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr 165 170 175 Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln 180 185 190 Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys 195 200 205 Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu 210 215 220 Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Leu Glu Glu Arg Asp Cys 225 230 235 240 Arg Val Ser Ser Phe Arg Val Lys Glu Asn Phe Asp Lys Ala Arg Phe 245 250 255 Ser Gly Thr Trp Tyr Ala Met Ala Lys Lys Asp Pro Glu Gly Leu Phe 260 265 270 Leu Gln Asp Asn Ile Val Ala Glu Phe Ser Val Asp Glu Thr Gly Gln 275 280 285 Met Ser Ala Thr Ala Lys Gly Arg Val Arg Leu Leu Asn Asn Trp Asp 290 295 300 Val Cys Ala Asp Met Val Gly Thr Phe Thr Asp Thr Glu Asp Pro Ala 305 310 315 320 Lys Phe Lys Met Lys Tyr Trp Gly Val Ala Ser Phe Leu Gln Lys Gly 325 330 335 Asn Asp Asp His Trp Ile Val Asp Thr Asp Tyr Asp Thr Tyr Ala Val 340 345 350 Gln Tyr Ser Cys Arg Leu Leu Asn Leu Asp Gly Thr Cys Ala Asp Ser 355 360 365 Tyr Ser Phe Val Phe Ser Arg Asp Pro Asn Gly Leu Pro Pro Glu Ala 370 375 380 Gln Lys Ile Val Arg Gln Arg Gln Glu Glu Leu Cys Leu Ala Arg Gln 385 390 395 400 Tyr Arg Leu Ile Val His Asn Gly Tyr Cys Asp Gly Arg Ser Glu Arg 405 410 415 Asn Leu Leu His His His His His His 420 425 12800PRTHomo sapiens 12Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu 290 295 300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 405 410 415 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 595 600 605 Leu Leu Glu Glu Arg Asp Cys Arg Val Ser Ser Phe Arg Val Lys Glu 610 615 620 Asn Phe Asp Lys Ala Arg Phe Ser Gly Thr Trp Tyr Ala Met Ala Lys 625 630 635 640 Lys Asp Pro Glu Gly Leu Phe Leu Gln Asp Asn Ile Val Ala Glu Phe 645 650 655 Ser Val Asp Glu Thr Gly Gln Met Ser Ala Thr Ala Lys Gly Arg Val 660 665 670 Arg Leu Leu Asn Asn Trp Asp Val Cys Ala Asp Met Val Gly Thr Phe 675 680 685 Thr Asp Thr Glu Asp Pro Ala Lys Phe Lys Met Lys Tyr Trp Gly Val 690 695 700 Ala Ser Phe Leu Gln Lys Gly Asn Asp Asp His Trp Ile Val Asp Thr 705 710 715 720 Asp Tyr Asp Thr Tyr Ala Val Gln Tyr Ser Cys Arg Leu Leu Asn Leu 725 730 735 Asp Gly Thr Cys Ala Asp Ser Tyr Ser Phe Val Phe Ser Arg Asp Pro 740 745 750 Asn Gly Leu Pro Pro Glu Ala Gln Lys Ile Val Arg Gln Arg Gln Glu 755 760 765 Glu Leu Cys Leu Ala Arg Gln Tyr Arg Leu Ile Val His Asn Gly Tyr 770 775 780 Cys Asp Gly Arg Ser Glu Arg Asn Leu Leu His His His His His His 785 790 795 800 13788PRTHomo sapiens 13Met Lys Trp Val Trp Ala Leu Leu Leu Leu Ala Ala Leu Gly Ser Gly 1 5 10 15 Arg Ala Glu Arg Asp Cys Arg Val Ser Ser Phe Arg Val Lys Glu Asn 20 25 30 Phe Asp Lys Ala Arg Phe Ser Gly Thr Trp Tyr Ala Met Ala Lys Lys 35 40 45 Asp Pro Glu Gly Leu Phe Leu Gln Asp Asn Ile Val Ala Glu Phe Ser 50 55 60 Val Asp Glu Thr Gly Gln Met Ser Ala Thr Ala Lys Gly Arg Val Arg 65 70 75 80 Leu Leu Asn Asn Trp Asp Val Cys Ala Asp Met Val Gly Thr Phe Thr 85 90 95 Asp Thr Glu Asp Pro Ala Lys Phe Lys Met Lys Tyr Trp Gly Val Ala 100 105 110 Ser Phe Leu Gln Lys Gly Asn Asp Asp His Trp Ile Val Asp Thr Asp 115 120 125 Tyr Asp Thr Tyr Ala Val Gln Tyr Ser Cys Arg Leu Leu Asn Leu Asp 130 135 140 Gly Thr Cys Ala Asp Ser Tyr Ser Phe Val Phe Ser Arg Asp Pro Asn 145 150 155 160 Gly Leu Pro Pro Glu Ala Gln Lys Ile Val Arg Gln Arg Gln Glu Glu 165 170 175 Leu Cys Leu Ala Arg Gln Tyr Arg Leu Ile Val His Asn Gly Tyr Cys 180 185 190 Asp Gly Arg Leu Glu Asp Ala His Lys Ser Glu Val Ala His Arg Phe 195 200 205 Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe 210 215 220 Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val 225 230 235 240 Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala 245 250 255 Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys 260 265 270 Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys 275 280 285 Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp 290 295 300 Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met 305 310 315 320 Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu 325 330 335 Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu 340 345 350 Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala 355 360 365 Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp 370 375 380 Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu 385 390 395 400 Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu 405 410 415 Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val 420 425 430 Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu 435 440 445 Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn 450 455 460 Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu 465 470 475 480 Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro 485 490 495 Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val 500 505 510 Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu 515 520 525 Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu 530 535 540 Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala 545 550 555 560 Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro 565 570 575 Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe 580 585 590 Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr 595 600 605 Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser 610 615 620 Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala 625 630 635 640 Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln 645 650 655 Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys 660 665 670 Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu 675 680 685 Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe 690 695 700 Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile 705 710 715 720 Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala 725 730 735 Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val 740 745 750 Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu 755 760 765 Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu His His 770 775 780 His His His His 785 141842DNAHomo sapiens 14atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60gtgtttcgtc gagatgcaca caagagtgag gttgctcatc ggtttaaaga tttgggagaa 120gaaaatttca aagccttggt gttgattgcc tttgctcagt atcttcagca gtgtccattt 180gaagatcatg taaaattagt gaatgaagta actgaatttg caaaaacatg tgttgctgat 240gagtcagctg aaaattgtga caaatcactt catacccttt ttggagacaa attatgcaca 300gttgcaactc ttcgtgaaac ctatggtgaa atggctgact gctgtgcaaa acaagaacct 360gagagaaatg aatgcttctt gcaacacaaa gatgacaacc caaacctccc ccgattggtg 420agaccagagg ttgatgtgat gtgcactgct tttcatgaca atgaagagac atttttgaaa 480aaatacttat atgaaattgc cagaagacat ccttactttt atgccccgga actccttttc 540tttgctaaaa ggtataaagc tgcttttaca gaatgttgcc aagctgctga taaagctgcc 600tgcctgttgc caaagctcga tgaacttcgg gatgaaggga aggcttcgtc tgccaaacag 660agactccaat tggagcgcga ctgccgagtg agcagcttcc gagtcaagga gaacttcgac 720aaggctcgct tctctgggac ctggtacgcc atggccaaga aggaccccga gggcctcttt 780ctgcaggaca acatcgtcgc ggagttctcc gtggacgaga ccggccagat gagcgccaca 840gccaagggcc gagtccgtct tttgaataac tgggacgtgt gcgcagacat ggtgggcacc 900ttcacagaca

ccgaggaccc tgccaagttc aagatgaagt actggggcgt agcctccttt 960ctccagaaag gaaatgatga ccactggatc gtcgacacag actacgacac gtatgccgtg 1020cagtactcct gccgcctcct gaacctcgat ggcacctgtg ctgacagcta ctccttcgtg 1080ttttcccggg accccaacgg cctgccccca gaagcgcaga agattgtaag gcagcggcag 1140gaggagctgt gcctggccag gcagtacagg ctgatcgtcc acaacggtta ctgcgatggc 1200agactcgagg aagagcctca gaatttaatc aaacaaaatt gtgagctttt tgagcagctt 1260ggagagtaca aattccagaa tgcgctatta gttcgttaca ccaagaaagt accccaagtg 1320tcaactccaa ctcttgtaga ggtctcaaga aacctaggaa aagtgggcag caaatgttgt 1380aaacatcctg aagcaaaaag aatgccctgt gcagaagact atctatccgt ggtcctgaac 1440cagttatgtg tgttgcatga gaaaacgcca gtaagtgaca gagtcaccaa atgctgcaca 1500gaatccttgg tgaacaggcg accatgcttt tcagctctgg aagtcgatga aacatacgtt 1560cccaaagagt ttaatgctga aacattcacc ttccatgcag atatatgcac actttctgag 1620aaggagagac aaatcaagaa acaaactgca cttgttgagc tcgtgaaaca caagcccaag 1680gcaacaaaag agcaactgaa agctgttatg gatgatttcg cagcttttgt agagaagtgc 1740tgcaaggctg acgataagga gacctgcttt gccgaggagg gtaaaaaact tgttgctgca 1800agtcaagctg ccttaggctt acatcatcat catcatcatt aa 1842151860DNAHomo sapiens 15atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60gtgtttcgtc gagatgcaca caagcaattg gaagagcctc agaatttaat caaacaaaat 120tgtgagcttt ttgagcagct tggagagtac aaattccaga atgcgctatt agttcgttac 180accaagaaag taccccaagt gtcaactcca actcttgtag aggtctcaag aaacctagga 240aaagtgggca gcaaatgttg taaacatcct gaagcaaaaa gaatgccctg tgcagaagac 300tatctatccg tggtcctgaa ccagttatgt gtgttgcatg agaaaacgcc agtaagtgac 360agagtcacca aatgctgcac agaatccttg gtgaacaggc gaccatgctt ttcagctctg 420gaagtcgatg aaacatacgt tcccaaagag tttaatgctg aaacattcac cttccatgca 480gatatatgca cactttctga gaaggagaga caaatcaaga aacaaactgc acttgttgag 540ctcgtgaaac acaagcccaa ggcaacaaaa gagcaactga aagctgttat ggatgatttc 600gcagcttttg tagagaagtg ctgcaaggct gacgataagg agacctgctt tgccgaggag 660ggtaaaaaac ttgttgctgc aagtcaagct gccttaggct tactcgagga gcgcgactgc 720cgagtgagca gcttccgagt caaggagaac ttcgacaagg ctcgcttctc tgggacctgg 780tacgccatgg ccaagaagga ccccgagggc ctctttctgc aggacaacat cgtcgcggag 840ttctccgtgg acgagaccgg ccagatgagc gccacagcca agggccgagt ccgtcttttg 900aataactggg acgtgtgcgc agacatggtg ggcaccttca cagacaccga ggaccctgcc 960aagttcaaga tgaagtactg gggcgtagcc tcctttctcc agaaaggaaa tgatgaccac 1020tggatcgtcg acacagacta cgacacgtat gccgtgcagt actcctgccg cctcctgaac 1080ctcgatggca cctgtgctga cagctactcc ttcgtgtttt cccgggaccc caacggcctg 1140cccccagaag cgcagaagat tgtaaggcag cggcaggagg agctgtgcct ggccaggcag 1200tacaggctga tcgtccacaa cggttactgc gatggcagat ctagagatgc acacaagagt 1260gaggttgctc atcggtttaa agatttggga gaagaaaatt tcaaagcctt ggtgttgatt 1320gcctttgctc agtatcttca gcagtgtcca tttgaagatc atgtaaaatt agtgaatgaa 1380gtaactgaat ttgcaaaaac atgtgttgct gatgagtcag ctgaaaattg tgacaaatca 1440cttcataccc tttttggaga caaattatgc acagttgcaa ctcttcgtga aacctatggt 1500gaaatggctg actgctgtgc aaaacaagaa cctgagagaa atgaatgctt cttgcaacac 1560aaagatgaca acccaaacct cccccgattg gtgagaccag aggttgatgt gatgtgcact 1620gcttttcatg acaatgaaga gacatttttg aaaaaatact tatatgaaat tgccagaaga 1680catccttact tttatgcccc ggaactcctt ttctttgcta aaaggtataa agctgctttt 1740acagaatgtt gccaagctgc tgataaagct gcctgcctgt tgccaaagct cgatgaactt 1800cgggatgaag ggaaggcttc gtctgccaaa cagagactcc atcatcatca tcatcattaa 1860161224DNAHomo sapiens 16atgaagtggg tgtgggcgct cttgctgttg gcggcgctgg gcagcggccg cgcggagcgc 60gactgccgag tgagcagctt ccgagtcaag gagaacttcg acaaggctcg cttctctggg 120acctggtacg ccatggccaa gaaggacccc gagggcctct ttctgcagga caacatcgtc 180gcggagttct ccgtggacga gaccggccag atgagcgcca cagccaaggg ccgagtccgt 240cttttgaata actgggacgt gtgcgcagac atggtgggca ccttcacaga caccgaggac 300cctgccaagt tcaagatgaa gtactggggc gtagcctcct ttctccagaa aggaaatgat 360gaccactgga tcgtcgacac agactacgac acgtatgccg tgcagtactc ctgccgcctc 420ctgaacctcg atggcacctg tgctgacagc tactccttcg tgttttcccg ggaccccaac 480ggcctgcccc cagaagcgca gaagattgta aggcagcggc aggaggagct gtgcctggcc 540aggcagtaca ggctgatcgt ccacaacggt tactgcgatg gcagactcga ggaagagcct 600cagaatttaa tcaaacaaaa ttgtgagctt tttgagcagc ttggagagta caaattccag 660aatgcgctat tagttcgtta caccaagaaa gtaccccaag tgtcaactcc aactcttgta 720gaggtctcaa gaaacctagg aaaagtgggc agcaaatgtt gtaaacatcc tgaagcaaaa 780agaatgccct gtgcagaaga ctatctatcc gtggtcctga accagttatg tgtgttgcat 840gagaaaacgc cagtaagtga cagagtcacc aaatgctgca cagaatcctt ggtgaacagg 900cgaccatgct tttcagctct ggaagtcgat gaaacatacg ttcccaaaga gtttaatgct 960gaaacattca ccttccatgc agatatatgc acactttctg agaaggagag acaaatcaag 1020aaacaaactg cacttgttga gctcgtgaaa cacaagccca aggcaacaaa agagcaactg 1080aaagctgtta tggatgattt cgcagctttt gtagagaagt gctgcaaggc tgacgataag 1140gagacctgct ttgccgagga gggtaaaaaa cttgttgctg caagtcaagc tgccttaggc 1200ttacatcatc atcatcatca ttaa 1224171278DNAHomo sapiens 17atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60gtgtttcgtc gagatgcaca caagcaattg gaagagcctc agaatttaat caaacaaaat 120tgtgagcttt ttgagcagct tggagagtac aaattccaga atgcgctatt agttcgttac 180accaagaaag taccccaagt gtcaactcca actcttgtag aggtctcaag aaacctagga 240aaagtgggca gcaaatgttg taaacatcct gaagcaaaaa gaatgccctg tgcagaagac 300tatctatccg tggtcctgaa ccagttatgt gtgttgcatg agaaaacgcc agtaagtgac 360agagtcacca aatgctgcac agaatccttg gtgaacaggc gaccatgctt ttcagctctg 420gaagtcgatg aaacatacgt tcccaaagag tttaatgctg aaacattcac cttccatgca 480gatatatgca cactttctga gaaggagaga caaatcaaga aacaaactgc acttgttgag 540ctcgtgaaac acaagcccaa ggcaacaaaa gagcaactga aagctgttat ggatgatttc 600gcagcttttg tagagaagtg ctgcaaggct gacgataagg agacctgctt tgccgaggag 660ggtaaaaaac ttgttgctgc aagtcaagct gccttaggct tactcgagga gcgcgactgc 720cgagtgagca gcttccgagt caaggagaac ttcgacaagg ctcgcttctc tgggacctgg 780tacgccatgg ccaagaagga ccccgagggc ctctttctgc aggacaacat cgtcgcggag 840ttctccgtgg acgagaccgg ccagatgagc gccacagcca agggccgagt ccgtcttttg 900aataactggg acgtgtgcgc agacatggtg ggcaccttca cagacaccga ggaccctgcc 960aagttcaaga tgaagtactg gggcgtagcc tcctttctcc agaaaggaaa tgatgaccac 1020tggatcgtcg acacagacta cgacacgtat gccgtgcagt actcctgccg cctcctgaac 1080ctcgatggca cctgtgctga cagctactcc ttcgtgtttt cccgggaccc caacggcctg 1140cccccagaag cgcagaagat tgtaaggcag cggcaggagg agctgtgcct ggccaggcag 1200tacaggctga tcgtccacaa cggttactgc gatggcagat cagaaagaaa ccttttgcat 1260catcatcatc atcattag 1278182403DNAHomo sapiens 18atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60gtgtttcgtc gagatgcaca caagagtgag gttgctcatc ggtttaaaga tttgggagaa 120gaaaatttca aagccttggt gttgattgcc tttgctcagt atcttcagca gtgtccattt 180gaagatcatg taaaattagt gaatgaagta actgaatttg caaaaacatg tgttgctgat 240gagtcagctg aaaattgtga caaatcactt catacccttt ttggagacaa attatgcaca 300gttgcaactc ttcgtgaaac ctatggtgaa atggctgact gctgtgcaaa acaagaacct 360gagagaaatg aatgcttctt gcaacacaaa gatgacaacc caaacctccc ccgattggtg 420agaccagagg ttgatgtgat gtgcactgct tttcatgaca atgaagagac atttttgaaa 480aaatacttat atgaaattgc cagaagacat ccttactttt atgccccgga actccttttc 540tttgctaaaa ggtataaagc tgcttttaca gaatgttgcc aagctgctga taaagctgcc 600tgcctgttgc caaagctcga tgaacttcgg gatgaaggga aggcttcgtc tgccaaacag 660agactcaagt gtgccagtct ccaaaaattt ggagaaagag ctttcaaagc atgggcagta 720gctcgcctga gccagagatt tcccaaagct gagtttgcag aagtttccaa gttagtgaca 780gatcttacca aagtccacac ggaatgctgc catggagatc tgcttgaatg tgctgatgac 840agggcggacc ttgccaagta tatctgtgaa aatcaagatt cgatctccag taaactgaag 900gaatgctgtg aaaaacctct gttggaaaaa tcccactgca ttgccgaagt ggaaaatgat 960gagatgcctg ctgacttgcc ttcattagct gctgattttg ttgaaagtaa ggatgtttgc 1020aaaaactatg ctgaggcaaa ggatgtcttc ctgggcatgt ttttgtatga atatgcaaga 1080aggcatcctg attactctgt cgtgctgctg ctgagacttg ccaagacata tgaaaccact 1140ctagagaagt gctgtgccgc tgcagatcct catgaatgct atgccaaagt gttcgatgaa 1200tttaaacctc ttgtggaaga gcctcagaat ttaatcaaac aaaattgtga gctttttgag 1260cagcttggag agtacaaatt ccagaatgcg ctattagttc gttacaccaa gaaagtaccc 1320caagtgtcaa ctccaactct tgtagaggtc tcaagaaacc taggaaaagt gggcagcaaa 1380tgttgtaaac atcctgaagc aaaaagaatg ccctgtgcag aagactatct atccgtggtc 1440ctgaaccagt tatgtgtgtt gcatgagaaa acgccagtaa gtgacagagt caccaaatgc 1500tgcacagaat ccttggtgaa caggcgacca tgcttttcag ctctggaagt cgatgaaaca 1560tacgttccca aagagtttaa tgctgaaaca ttcaccttcc atgcagatat atgcacactt 1620tctgagaagg agagacaaat caagaaacaa actgcacttg ttgagctcgt gaaacacaag 1680cccaaggcaa caaaagagca actgaaagct gttatggatg atttcgcagc ttttgtagag 1740aagtgctgca aggctgacga taaggagacc tgctttgccg aggagggtaa aaaacttgtt 1800gctgcaagtc aagctgcctt aggcttactc gaggagcgcg actgccgagt gagcagcttc 1860cgagtcaagg agaacttcga caaggctcgc ttctctggga cctggtacgc catggccaag 1920aaggaccccg agggcctctt tctgcaggac aacatcgtcg cggagttctc cgtggacgag 1980accggccaga tgagcgccac agccaagggc cgagtccgtc ttttgaataa ctgggacgtg 2040tgcgcagaca tggtgggcac cttcacagac accgaggacc ctgccaagtt caagatgaag 2100tactggggcg tagcctcctt tctccagaaa ggaaatgatg accactggat cgtcgacaca 2160gactacgaca cgtatgccgt gcagtactcc tgccgcctcc tgaacctcga tggcacctgt 2220gctgacagct actccttcgt gttttcccgg gaccccaacg gcctgccccc agaagcgcag 2280aagattgtaa ggcagcggca ggaggagctg tgcctggcca ggcagtacag gctgatcgtc 2340cacaacggtt actgcgatgg cagatcagaa agaaaccttt tgcatcatca tcatcatcat 2400tag 2403192367DNAHomo sapiens 19atgaagtggg tgtgggcgct cttgctgttg gcggcgctgg gcagcggccg cgcggagcgc 60gactgccgag tgagcagctt ccgagtcaag gagaacttcg acaaggctcg cttctctggg 120acctggtacg ccatggccaa gaaggacccc gagggcctct ttctgcagga caacatcgtc 180gcggagttct ccgtggacga gaccggccag atgagcgcca cagccaaggg ccgagtccgt 240cttttgaata actgggacgt gtgcgcagac atggtgggca ccttcacaga caccgaggac 300cctgccaagt tcaagatgaa gtactggggc gtagcctcct ttctccagaa aggaaatgat 360gaccactgga tcgtcgacac agactacgac acgtatgccg tgcagtactc ctgccgcctc 420ctgaacctcg atggcacctg tgctgacagc tactccttcg tgttttcccg ggaccccaac 480ggcctgcccc cagaagcgca gaagattgta aggcagcggc aggaggagct gtgcctggcc 540aggcagtaca ggctgatcgt ccacaacggt tactgcgatg gcagactcga ggatgcacac 600aagagtgagg ttgctcatcg gtttaaagat ttgggagaag aaaatttcaa agccttggtg 660ttgattgcct ttgctcagta tcttcagcag tgtccatttg aagatcatgt aaaattagtg 720aatgaagtaa ctgaatttgc aaaaacatgt gttgctgatg agtcagctga aaattgtgac 780aaatcacttc ataccctttt tggagacaaa ttatgcacag ttgcaactct tcgtgaaacc 840tatggtgaaa tggctgactg ctgtgcaaaa caagaacctg agagaaatga atgcttcttg 900caacacaaag atgacaaccc aaacctcccc cgattggtga gaccagaggt tgatgtgatg 960tgcactgctt ttcatgacaa tgaagagaca tttttgaaaa aatacttata tgaaattgcc 1020agaagacatc cttactttta tgccccggaa ctccttttct ttgctaaaag gtataaagct 1080gcttttacag aatgttgcca agctgctgat aaagctgcct gcctgttgcc aaagctcgat 1140gaacttcggg atgaagggaa ggcttcgtct gccaaacaga gactcaagtg tgccagtctc 1200caaaaatttg gagaaagagc tttcaaagca tgggcagtag ctcgcctgag ccagagattt 1260cccaaagctg agtttgcaga agtttccaag ttagtgacag atcttaccaa agtccacacg 1320gaatgctgcc atggagatct gcttgaatgt gctgatgaca gggcggacct tgccaagtat 1380atctgtgaaa atcaagattc gatctccagt aaactgaagg aatgctgtga aaaacctctg 1440ttggaaaaat cccactgcat tgccgaagtg gaaaatgatg agatgcctgc tgacttgcct 1500tcattagctg ctgattttgt tgaaagtaag gatgtttgca aaaactatgc tgaggcaaag 1560gatgtcttcc tgggcatgtt tttgtatgaa tatgcaagaa ggcatcctga ttactctgtc 1620gtgctgctgc tgagacttgc caagacatat gaaaccactc tagagaagtg ctgtgccgct 1680gcagatcctc atgaatgcta tgccaaagtg ttcgatgaat ttaaacctct tgtggaagag 1740cctcagaatt taatcaaaca aaattgtgag ctttttgagc agcttggaga gtacaaattc 1800cagaatgcgc tattagttcg ttacaccaag aaagtacccc aagtgtcaac tccaactctt 1860gtagaggtct caagaaacct aggaaaagtg ggcagcaaat gttgtaaaca tcctgaagca 1920aaaagaatgc cctgtgcaga agactatcta tccgtggtcc tgaaccagtt atgtgtgttg 1980catgagaaaa cgccagtaag tgacagagtc accaaatgct gcacagaatc cttggtgaac 2040aggcgaccat gcttttcagc tctggaagtc gatgaaacat acgttcccaa agagtttaat 2100gctgaaacat tcaccttcca tgcagatata tgcacacttt ctgagaagga gagacaaatc 2160aagaaacaaa ctgcacttgt tgagctcgtg aaacacaagc ccaaggcaac aaaagagcaa 2220ctgaaagctg ttatggatga tttcgcagct tttgtagaga agtgctgcaa ggctgacgat 2280aaggagacct gctttgccga ggagggtaaa aaacttgttg ctgcaagtca agctgcctta 2340ggcttacatc atcatcatca tcattaa 23672032DNAArtificial Sequencesense primer for albumin domain I in rat 20ggggtacccc accatgaagt gggtaacctt tc 322127DNAArtificial Sequenceantisense primer for albumin domain I in rat 21ccccaattgc atcctctgac ggacagc 272227DNAArtificial Sequencesense primer for RBP(55-585) in rat 22gggcaattgg agcgcgactg cagggtg 272327DNAArtificial Sequenceantisense primer for RBP(55-585) in rat 23cccctcgagt ctgctttgac agtaacc 272427DNAArtificial Sequencesense primer for albumin domain III in rat 24gggctcgagg aagaacctaa gaacttg 272548DNAArtificial Sequenceantisense primer for albumin domain III in rat 25ggctctagat taatgatgat gatgatgatg ggctaaggct tctttgct 482630DNAArtificial Sequencesense primer for RBP(1-585) in rat 26gcggaattcc accatggagt gggtgtgggc 302727DNAArtificial Sequenceantisense primer for RBP(1-585) in rat 27cccctcgagt ctgctttgac agtaacc 272832DNAArtificial Sequencesense primer for albumin domain I in mouse 28ggggtacccc accatgaagt gggtaacctt tc 322927DNAArtificial Sequenceantisense primer for albumin domain I in mouse 29ccccaattgc attctctgac ggacaga 273027DNAArtificial Sequencesense primer for albumin domain III in mouse 30gggctcgagg aagagcctaa gaacttg 273148DNAArtificial Sequenceantisense primer for albumin domain III in mouse 31ggctctagat taatgatgat gatgatgatg ggctaaggtg tctttgca 483230DNAArtificial Sequencesense primer for RBP(1-585) in mouse 32gcggaattcc accatggagt gggtgtgggc 303327DNAArtificial Sequenceantisense primer for RBP(1-585) in mouse 33cccctcgagc ctgctttgac agtaacc 273427DNAArtificial Sequencesense primer for RBP(55-585) in mouse 34gggcaattgg agcgcgactg cagggtg 273527DNAArtificial Sequenceantisense primer for RBP(55-585) in mouse 35cccctcgagc ctgctttgac agtaacc 27