Recombinant antigens for diagnosis and prevention of spotted fever rickettsiae

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates to genes and proteins that can be used in vaccination, detection and identification of spotted fever rickettsiae infection. More particularly, the invention relates to specific nucleotide sequences encoding a highly specific and immunogenic portion of the outer membrane protein A (OmpA) of Rickettsia rickettsii. The polypeptide sequence can be utilized in diagnostic and detection assays for spotted fever rickettsiae and as an immunogen component in vaccine formulations against spotted fever Rickettsia.

2. Description of the Prior Art

Rickettsiae are Gram-negative, obligate intracellular parasites of the genus Rickettsia. They are responsible for many spotted fever group Rickettsia infection, and typhus group infection [1]. Of the spotted fever group, Rocky Mountain spotted fever is the most severe and most frequently reported rickettsial illness in the United States. It also occurs in Mexico and in central and south America. The disease is caused by Rickettsia rickettsii, a species of bacteria that is transmitted to humans by ixodid (hard) ticks. Rickettsiae target vascular endothelium. Other named species of the spotted fever group and their geographic distributions are listed in table 1.

Table 1 of Human Disease around the world caused by spotted fever group Rickettsiae. (http://www.cdc.gov/ncidod/dvrd/rmsf/Epidemiology.html, Apr. 1, 2008).

TABLE-US-00001 Organism Disease or Presentation Geographic Location Rickettsia rickettsii Rocky Mountain spotted fever North, Central and South America Rickettsia conorii Mediterranean spotted fever, Europe, Asia, Africa, India, boutonneuse fever, Israeli spotted fever, Israel, Sicily, Russia Astrakhan fever, Indian tick typhus Rickettsia akari Rickettsialpox Worldwide Rickettsia sibirica Siberian tick typhus, North Asian tick Siberia, People's Republic of typhus China, Mongolia, Europe Rickettsia australis Queensland tick typhus Australia Rickettsia honei Flinders Island spotted fever, Thai tick Australia, South Eastern Asia typhus Rickettsia africae African tick-bite fever Sub Saharan Africa, Caribbean Rickettsia japonica Japanese or Oriental spotted fever Japan Rickettsia felis Cat flea rickettsiosis, flea borne typhus Worldwide Rickettsia slovaca Necrosis, erythema, lymphoadenopathy Europe Rickettsia Mild spotted fever China, Asian Region of heilongjaiangensis Russia Rickettsia parkeri Mild spotted fever USA

Rocky Mountain spotted fever (RMSF) can be treated easily with antibiotics. If the patient is treated within the first 4-5 days of the disease, fever generally subsides within 24-72 hours. However, it is very difficult to make a clinical diagnosis in the disease's early stages. The early clinical presentation of Rocky Mountain spotted fever is nonspecific and may resemble a variety of other diseases including influenza, measles, and rubella, as well as other rickettsial diseases [2]. The classic symptoms of RMSF are sudden onset of fever, headache, and muscle pain, followed by development of rash, abdominal pain, joint pain, and diarrhea [2]. However, this combination of symptoms is not always detected when the patient initially presents for care. Without prompt and appropriate treatment, the disease can be fatal. Long-term health problems following acute Rocky Mountain spotted fever infection include partial paralysis of the lower extremities, gangrene requiring amputation of fingers, toes, or arms or legs, hearing loss, loss of bowel or bladder control, movement disorders, and language disorders. These complications are most frequent in persons recovering from severe, life-threatening form of the disease, often following lengthy hospitalizations. Therefore, rapid and accurate diagnosis and treatment at the onset of the disease is desirable.

Serologic assays are the most widely available and frequently used methods for confirming cases of Rocky Mountain spotted fever. The indirect immunofluoresence assay (IFA) is generally considered the reference standard in Rocky Mountain spotted fever serology and is the test currently used by Center of Disease Control and most state public health laboratories, but other well validated assays can also be used in diagnosis, including indirect hemagglutination assay (IHA), ELISA, latex agglutination, and dot immunoassays [2].

IFA can be used to detect either IgG or IgM antibodies. Blood samples taken at early (acute) and late (convalescent) stages of the disease are the preferred specimens for evaluation. Most patients demonstrate increased IgM titers by the end of the first week of illness. Diagnostic levels of IgG antibody generally do not appear until 7-10 days after the onset of illness. The value of testing two sequential serum or plasma samples together to show a rising antibody level is very important in confirming acute infection with rickettsial agents because antibody titers may persist in some individuals for years after the original exposure to cross-reactive rickettsial agents. IgG antibodies are more specific and reliable since other bacterial infections can also cause elevations in riskettsial IgM antibody titers. One of the disadvantage in using IFA to diagnose Rocky Mountain spotted fever is that most patients visit their physician relatively early in the course of the illness, before the presences of diagnostic antibody level. In addition, variations in the endpoint titer may occur due to differences in the quality of the microscope, the quality of the anti-immunoglobulin conjugate, and the experience of the technician.

Both IHA and latex agglutination rely on a common source of rickettsial antigen, extracted from R. rickettsii. This antigenic material is coated on sheep or human type O erythrocytes for IHA and onto latex beads for latex agglutination. Although IHA test demonstrates the earliest, steepest rise in antibody titer of all serologic tests for RMSF, it is rarely used as the diagnostic method in acute state of the illness. Only 19% of patients with RMSF had an acute titer of 40, which is much lower value than CDC's criterion for using a single titer indicating a probable diagnosis (.gtoreq.128) [2]. The latex agglutination test is technically simple and rapid and requires no elaborate equipment. However, latex agglutination test is inappropriate for serosurveys and is more diagnostically discriminatory for establishing the diagnosis of a recent infection because the detectable antibody has persistent presentation months after the onset of illness [2].

Although routinely used in retrospective confirmatory diagnosis, current serologic methods are not considered appropriate rapid acute diagnostic tests. Very seldom are specific antibodies to R. rickettsii detected during the acute stage of illness when empiric treatment must begin. The most rapid and specific diagnostic assays for Rocky Mountain spotted fever rely on molecular methods like PCR, which can detect DNA of 5-10 rickettsial organisms in a sample. While organisms can be detected in whole blood samples obtained at the acute onset of illness in a few hours, rickettsial DNA is most readily detected in fresh skin biopsies like those used in immunostaining procedures. PCR can also be done on the fixed tissues used in immunostaining, but it is less sensitive than with unfixed tissues. PCR methods can be R. rickettsii-specific but are usually confirmed by DNA sequencing of the amplified gene fragments. Consequently, this procedure is more specific than antibody-based methods which are often only genus or spotted fever group-specific. However, gene amplification requires sophisticated instrumentation and reagents generally not available in most rural medical facilities. In addition, extensive training is required for the end users to achieve accurate and standardized results.

Another approach to Rocky Mountain spotted fever diagnostics is immunostaining. This method is used by taking a skin biopsy of the rash from an infected patient prior to therapy or within the first 48 hours after starting the antibiotic therapy. However, because rickettsiae are focally distributed in lesions of Rocky Mountain spotted fever, this test may not always detect an agent. Even in laboratories with expertise in performing this test, the sensitivity is only about 70% on biopsied tissues because of the scarcity of organisms in some samples.

Two major outer membrane proteins of spotted fever group Rickettsiae, OmpA and OmpB, have been identified as major immunogenic antigens. Outer membrane protein A (OmpA) has an apparent molecular mass of 190 kDa. Immunization with recombinant rOmpA (Rickettsia OmpA) protects guinea pigs against a lethal dose of R. rickettsii. The rOmpA gene of (R. Rickettsia) contains 6747 nucleotides that code for 2249 amino acid protein [5]. Moreover, immunization with recombinant OmpA of R. conorii completely protects guinea pigs against challenge with R. conorii and partially protects against challenge with R. rickettsii [6]. OmpA of R. Rickettsii was also shown to react strongly with sera from patients infected with R. rickettsii and other spotted fever group Rickettsia. Based on these observations, therefore, OmpA is a particularly advantageous target for developing diagnostic reagents against spotted fever group Rickettsia, especially against R. Rickettsii, the causative agent of RMSF.

FIG. 1 shows that fragment X of Omp A is conserved among various spotted fever group Rickettsia. FIG. 2 is a comparison of fragment Y of OmpA from various spotted fever group Rickettsia.

SUMMARY OF INVENTION

Accordingly, an object of this invention is recombinant polypeptides encompassing immunologic regions of OmpA of R. Rickettsii.

Another object of the invention is a method for using recombinant polypeptides encompassing immunologic regions of OmpA of R. Rickettsii in antibody-based assays for the detection of spotted fever group rickettsiae.

Yet another object of the invention is a method for using recombinant polypeptides encompassing immunologic regions of OmpA R. Rickettsii in vaccine formulations against spotted fever infections.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows alignment of polypeptide sequences of X fragment of the OmpA ("OmpA_X") from various spotted fever group Rickettsia, including R. slovaca (SEQ ID NO: 13), R. honei (SEQ ID NO:14), R. rickettsii (SEQ ID NO: 3), R. peacockii (SEQ ID NO:16), R. parkeri (SEQ ID NO: 17).

FIG. 2 shows alignment of polypeptide sequences of the Y fragment of the OmpA ("OmpA_Y") from various spotted fever group Rickettsia, R. conorii (SEQ ID NO:19), R. slovaca (SEQ ID NO:20), R. honei (SEQ ID NO: 18), R. rickettsii (SEQ ID NO:5), R. parkeri (SEQ ID NO:21).

FIG. 3 Open reading frame of OmpA and location of Fragments X, Y and Z.

FIG. 4 Vector maps for (a) pET24a and (b) pET28a.

FIG. 5A Western blot analysis of OmpA, OmpB, and Fragment X with normal serum.

FIG. 5B Western blot analysis of OmpA, OmpB, Fragment X with patient serum.

FIG. 6A Western blot analysis of OmpA, OmpB, and Fragment Y with normal serum.

FIG. 6B Western blot analysis of OmpA, OmpB, and Fragment Y with patient serum.

FIG. 7 Western blot analysis of Fragment Z with normal (a) serum, and patient serum (b).

FIG. 8 Prototype SFG test assay made with OmpA-X and Y proteins.

DETAILED DESCRIPTION OF THE INVENTION

Study of spotted fever group Rickettsia has led to the identification of OmpA, which is an exceptionally promising candidate as a reagent for use in diagnostic and detection assays, as well as in vaccine formulations. Central to the development and standardization of improved detection and diagnostic immunoassay methods is the development of more effective antigens for use in antibody-based methods. With a molecular weight of 190 kDa, however, OmpA is difficult to express and purify. Therefore, the large OmpA (190 kDa), is divided into five epitope-containing fragments: W (94 kDa), R (47 kDa), X (25 kDa), Y (26 kDa), and Z (70 kDa) (FIG. 3). Each fragment can be found in a region of OmpA that is predicted to be rich in epitopes. An epitope is the region of an antigen to which the variable region of an antibody binds. Most antigens have a large number of epitopes. The purified antigens may then be used to detect antibodies against Rickettsiae caused spotted fever, and diagnose the disease. By invoking an immunogenic reaction, the antigens may also be used as vaccines. FIG. 3 illustrates the location of these fragments within the OmpA molecule. The amino acid sequence of OmpA is illustrated in SEQ ID No. 1, which is encoded by nucleotide sequence set forth in SEQ ID No. 2. Fragment X has the amino acid sequence of SEQ ID No. 3 and is encoded by nucleotide sequence set forth in SEQ ID. No. 4. Fragment Y has the amino acid sequence of SEQ ID No. 5 and is encoded by nucleotide sequence set forth in SEQ ID 6. Fragment Z is a longer fragment containing both fragment X and Y. Fragment Z's amino acid sequence is set forth in in SEQ ID 11 and is encoded by nucleotide sequence set forth in SEQ ID. 12. Rapid tests made with purified antigen for R. rickettsii may enable timely, accurate diagnosis of Rocky Mountain spotted fever, which can be performed even in locations where laboratory equipment is not available.

Construction of recombinant R. rickettisia OmpA fragments were carried out by the expression, purification, and refolding of the X, Y and Z fragments (OmpA-X, OmpA-Y and OmpA-Z). The gene coding OmpA-X (from a.a. 1281 to a.a. 1522 of OmpA) was cloned into the expression vector pET24a. The gene encoding OmpA-Y (from amino acids 1700 to 1950 of OmpA) was cloned into the expression vector pET28a. The gene encoding OmpA-Z (from amino acids 1281 to 1950) was cloned into the expression vector pET 24a. FIG. 4 shows the Vector maps for (a) pET24a and (b) pET28a. BL21(DE3) strain of Escherichia coli was transformed with plasmid containing gene segments for OmpA-X, OmpA-Y and Omp-Z. Following induction, the cells were lysed, and OmpA-X, OmpA-Y and Omp-Z was each found in the inclusion body. The solubilized OmpA-X, OmpA-Y and OmpA-Z in 8 M urea was purified by His-tag affinity chromatography. SDS-PAGE demonstrated that greater than 90% purity was achieved in the final elution. The purified protein fragments were refolded by sequential dialysis at 4.degree. C. in progressively lower concentrations of urea. N-terminal protein sequencing was used to confirm the identity of the refolded proteins. Western blot experiments showed that OmpA-X, OmpA-Y and Omp-Z reacted with patient sera, OmpA-Y reacted especially strong, suggesting that these fragments can be used to develop rapid sero-diagnostic assays and may even be a candidate for a vaccine against Rocky Mountain spotted fever.

Cloning of OmpA X, Y and Z Genes into Expression Vectors

A set of oligonucleotide primers were used to amplify X and Y fragments.

TABLE-US-00002 TABLE 2 Primer sets for OmpA-X, OmpA-Y and OmpA-Z fragments. SEQ ID No. 7 OmAXf GGT GGT CAT ATG CGA GAT TCT GTT TTA GTA CTT TCT SEQ ID No. 8 OmAXr GGT GGT CTC GAG TTG AGT TAA TTG AAC AGC ATC ATT A SEQ ID No. 9 OmAYf GGT GGT CAT ATG GTA ACG GCT ACC AGC TTT GTA G SEQ ID No. 10 OmAYr GGT GGT CTC GAG TGA TTG TAC TTT ATC CAT TCT AGC SEQ ID No. 7 OmAZf GGT GGT CAT ATG CGA GAT TGT GTT TTA GTA CTT TCT SEQ ID No. 10 OmAZr GGT GGT CTC GAG TGA TTG TAC TTT ATC CAT TCT AGC

The expression vector pET24a and pET28a were digested with NdeI and XhoI (NEW ENGLAND BIOLABS.RTM. Inc, Beverly Mass.). The PCR product for OmpA-X, OmpA-Y and Omp-Z were also digested with NdeI and XhoI. Agarose gel electrophoresis was performed with the samples. The digested vectors and the X and Y inserts were then cut out from the gel and purified using the QIAquick Gel Extraction Kit (QIAGEN.TM., Valencia Calif.). The vectors and inserts were ligated together with T4DNA ligase (INVITROGEN.TM., Carlsbad Calif.). E. coli TOPO10 was transform with the resulting plasmids, now pET24a-OmpA-X, pET24a-OmpA-Z and pET28a-OmpA-Y for cloning. The transformed cells were spread on agar plates that contained 50 .mu.g/mL kanamycin and incubated overnight at 37.degree. C. The plasmids were then purified using QIAprep Spin Miniprep Kit (QIAGEN.TM., Valencia Calif.). A sample of the purified DNA was subjected to a double digestion with NdeI and XhoI, and agarose gel electrophoresis was performed to verify that the inserts had been successfully ligated into the vectors. The pET28a-OmpA-Y plasmid encoded OmpA-Y with a histidine tag on both the N- and C-terminuses, and the pET24a-OmpA-X plasmid encoded OmpA-X with a histidine tag on the C-terminus. The pET24a-OmpA-Z plasmid encoded OmpA-Z with a histidine tag on the C-terminus

Expression of OmpA-X, OmpA-Y and OmpA-Z

The plasmids containing the insert were used to transform into the expression hosts. The recombinant E. coli were spread on agar plates containing 50 .mu.g/mL kanamycin and were incubated at 37.degree. C. overnight. The recombinant E. coli expressing the OmpA fragment was then grown in 0.5 L OVERNIGHT EXPRESS.TM. Instant TB Medium (NOVAGEN.RTM., Gibbstown N.J.) containing 50 .mu.g/mL kanamycin at 37.degree. C. with shaking at room temperature. The medium induced protein expression. Following induction, the cells were harvested by centrifugation. The cell pellets were resuspended in 15 mL BUGBUSTER.RTM. Master Mix (NOVAGEN.RTM., Gibbstown N.J.). The lysis reaction was allowed to proceed for 30 minutes with shaking. The cells were also subjected to ultrasonic disruption on a sonicator for further lysing. The cell lysate was then centrifuged at 7400.times.g for 30 minutes. The supernatant was collected and saved. The pellet was resuspended in 30 mL 1.times.HisBind buffer (20 mM Tris-Hcl pH 8, 0.5 M NaCl, 10 mM imidazole) and centrifuged for 30 minutes as before. The supernatant was collected and saved. This process was repeated with 30 mL 1.times.HisBind buffer containing 2M urea, and finally with 15 mL 1.times.HisBind buffer containing 8M urea. Bradford's test and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the fractions and determine the protein concentration of OmpA-X, OmpA-Y and OmpA-Z.

Purification of OmpA-X and OmpA-Y

Ni-NTA His-bind Resin (NOVAGEN.RTM., Gibbstown N.J.) was used to purify OmpA fragments from the impurities in the sample. Two milliliters of the Ni-NTA His-bind resin suspension were added to make a 1 mL column. The column was equilibrated with 10 mL of 1.times.HisBind buffer containing 8M urea. Both OmpA-X and OmpA-Y were found in the inclusion body. The solubilized protein in 8M urea was then added to the column. After an incubation period of 30 minutes with shaking, the resin was allowed to settle. The flow-through was collected and saved. The column was washed twice with 10 mL 1.times.HisBind buffer containing 8 M urea. OmpA fragment was eluted with six 1 mL solutions, each with an increasing concentration of imidazole (20 mM, 50 mM, 100 mM, 200 mM, 400 mM, and 600 mM). Bradford's test was performed to determine the protein concentration of each fraction, and SDS-PAGE was used to analyze the purity of the samples.

Refolding of OmpA-X and OmpA-Y

The purified OmpA fragments in 8M urea was refolded by dialysis against progressively lower concentrations of urea (6 M, 4 M, 2 M, 1 M, 0 M, and 0 M) at 4.degree. C. In addition to urea, each buffer contained 20 mM Tris-HCl (pH 8), 0.15 M NaCl, 1 mM dithiothreitol (DTT), and 1 mM ethylenediamine tetraacetic acid (EDTA). The D-Tube.TM. Dialyzer Maxi MWCO 12-14 kDa (NOVAGEN.RTM., Gibbstown N.J.) was used for dialysis. The tube was hydrated for 30 seconds in 6M urea buffer, and then the protein sample to be refolded was added to the tube. The beaker containing the buffer and the tubes was placed on a stirrer for 1.5 hours. After this period of time, the tubes were transferred to the 4M urea buffer and left on the stirrer for 1.5 hours. This process was repeated for the rest of the buffers. After the second 0M urea buffer, the protein was recovered. Bradford's test was used to determine the protein concentration of the refolded sample.

Alternative Extraction, Purification and Refolding Procedures

To obtain OmpA fragments at a higher purity, alternative extraction, purification and refolding protocols may also be used. In this alternative embodiment, the cell pellet was well dispersed in buffer containing 20 mM Tris-HCl (pH 8), 1 mM dithiothreitol (DTT), and 1 mM ethylenediamine tetraacetic acid (EDTA), via up-down suction using a 10-ml pipette. The cells were broken using a Microfluidizer and centrifuged at 11,250.times.g for 30 min at 4.degree. C. Supernatant was disgarded and 2M urea in buffer containing 20 mM Tris-HCl (pH 8), 1 mM dithiothreitol (DTT), and 1 mM ethylenediamine tetraacetic acid (EDTA) was added to the pellet. The pellets were dispensed well and spin down as described before. These steps were repeated once. The supernatant was disgarded and 6 M urea in buffer containing 20 mM Tris-HCl (pH 8), 1 mM dithiothreitol (DTT), and 1 mM ethylenediamine tetraacetic acid (EDTA) was added. The pellet was well dispersed by up-down suction using a 10-ml pipette. The solution was centrifuged at 11,250.times.g for 30 min at 4.degree. C. Supernatant is collected for gel filtration purification.

Chromatographic purification of OmpA-X and OmpA-Y A were performed using gel filtration HPLC on TSK P3000SW (21.5 mm.times.30 cm) or TSK P4000SW (21.5 mm.times.60 cm) with Waters 600E and Phamarcia Recorder and monitor using a running buffer of 6 M urea, 20 mM Tris-HCl, 1 mM DTT, 1 mM EDTA, 0.15 M NaCl (pH8.0). Pre-equilibration of the TSK P3000SW in tandem with 4000SW preparative column by running the elution buffer on the column overnight (about 15 h) at a flow rate of 0.35 ml/min for 21.5 mm.times.30 cm column bed (about 100 ml) or at 0.65 ml/min for 21.5.times.60 cm column bed (208 ml). Isocratic elution run and fractionation were conducted on the sample with 6M urea solution loaded into a 5 ml loop at a flow rate of 4 ml/min. Fractionations were collected at the beginning of peak appearance and analyzed by gel electrophoresis for purity check.

Ni-NTA purification were then performed using Hisbind buffer containing 20 mM TrisHCl, 0.5 M NaCl, 10 mM imidazole, 8M Urea (pH 8.0). Fractions containing fragments of higher purity were pooled, and dialyzed for 30 minutes against 8M Urea in buffer containing 20 mM Tris-HCl (pH 8), 1 mM dithiothreitol (DTT), and 1 mM ethylenediamine tetraacetic acid (EDTA) and 0.5M NaCl twice at room temperature. The volume ratio of sample to buffer is 0.02 (i.e. 1 ml sample 50 ml buffer). Sample was loaded onto 1 ml of Ni-NTA (NOVAGEN.RTM., Gibbstown N.J.) column equilibrated with 10 ml Hisbind buffer. The sample was allowed the binding to perform for 15 min at RT with rocking. Collected flow-through samples and washed column with 20 ml Hisbind buffer. Proteins were then eluted with 6.times.1.0 ml of Hisbind buffer containing 25 mM, 50 mM, 100 mM, 200 mM, 500 mM, and 1M imidazole. Major protein appeared in fractions containing 50 to 200 mM imidazole. Check the elution purity in 4-20% SDS gel.

Chromatographic purification of OmpA-Z was performed in 6M urea buffer using an Anion-exchange HPLC on a Waters preparative DEAE column (21.5.times.15 cm, max. binding capacity, approx 700 mg protein) with Waters 2196 HPLC system interfaced with Millenium software. The starting buffer is 6M urea in 20 mM Tris-HCl (pH 8.0). The second buffer is 6 M urea in 0.5 M NaCl in 20 mM Tris-HCl (pH 8.0). The ending buffer is 6 M urea in 2 M NaCl in 20 mM Tris-HCl (pH 8.0). All buffers also contain 1 mM DTT, 1 mM EDTA, 20 M Tris-HCl (pH 7.5, Fisher). Pre-washing and equilibration were performed on DEAE column followed by fractionation at 0.15 min per fraction. Run SDS-4-20% Tris-HCl bio-rad ready gel BIO-RAD.RTM. Laboratories, Hercules, Calif.) to check the fractions. Poll the relative pure fractions for Ni-NTA column purification.

Ni-NTA column purification was conducted using HISBIND.RTM. buffer containing 20 mM TrisHCl, pH 8.0, 0.5 M NaCl, 10 mM imidazole, 8M Urea. Fractions containing fragments of higher purity were pooled and dialyzed for 30 minutes against 8M Urea in buffer containing 20 mM Tris-HCl (pH 8), 1 mM dithiothreitol (DTT), 1 mM ethylenediamine tetraacetic acid (EDTA) and 0.5M NaCl in Tris buffer pH8.0 twice at room temperature. The volume ratio of sample to buffer is 0.02 (i.e. 1 ml sample 50 ml buffer). Dialyzed sample was mixed with 1 ml of Ni-NTA (NOVAGEN.RTM., Gibbstown N.J.) column previously equilibrated with 10 ml Hisbind buffer. The mixture was rocking at room temperature for 15 minutes. The flow-through samples were then collected and washed column with 20 ml Hisbind buffer. Proteins were eluted with 6.times.1.0 ml of Hisbind buffer containing 25 mM, 50 mM, 100 mM, 200 mM, 400 mM, and 1M imidazole. The majority of OmpA-Z appeared in fractions containing 50 to 200 mM imidazole.

The purified OmpA polypeptide fragments (OmpA-X, OmpA-Y and OmpA-Z) were then refolded by dialysis against progressively lower concentrations of urea (4M, 2M, 1 M, and 0 M) at 4.degree. C. in buffer containing 20 mM Tris-HCl (pH 8), 0.15 M NaCl, 1 mM DTT, and 1 mM EDTA (900 ml). The sample was dialyzed against 4 M urea for 30 minutes in buffer containing 20 mM Tris-HCl (pH 8), 1 mM dithiothreitol (DTT), and 1 mM ethylenediamine tetraacetic acid (EDTA) twice in cold room. The volume ratio of sample to buffer is 0.05 (i.e. 1 ml sample 20 ml buffer). After dialysis in 0 M urea buffer, the protein was recovered and analyzed by BioRad protein assay and by SDS gel electrophoresis.

Example 1

Western Blot Experiment Demonstrating Immunogenic Reactivity of Fragments X, Y and Z

In order to ascertain the reactivity of the recombinant Fragments X, Y and Z as immunogenic antigens for Rocky Mountain spotted fever, western blot analysis was performed on Fragments X, Y and Z of OmpA of R. Rickettisia using normal sera and sera from patient who are known to be spotted fever positive. The purified and refolded proteins were subjected to SDS-PAGE and then electroblotted onto a nitrocellulose membrane (BIO-RAD.RTM. Laboratories, Hercules, Calif.). The entire procedure was carried out at room temperature. The membrane was blocked for non-specific binding by incubating with 10% skim milk in 1.times.TBS buffer for 1 hour with rocking. The milk was then poured off, and the membrane was washed once with 1.times.TBST buffer. Patient sera containing the primary antibody was diluted 100-fold in 1.times.TBST containing 5% milk. The membrane was incubated with the solution for 1 hour with rocking. Next, the membrane was washed three times with 1.times.TBST for ten minutes each, and the secondary antibody (goat anti-human IgG Horseradish Peroxidase Conjugate, 1:4000 dilution in 1.times.TBST). After 1 hour, the membrane was washed with 1.times.TBST. Three milliliters of OPTI-4CN Substrate were added to detect antibodies. After 10 minutes, the membrane was washed with water. FIG. 5 illustrates the specificity of the recombinant Fragment X by western blot analysis. In FIG. 5 (a), no reactivity was observed against OmpA, OmpB or Fragment X using control sera. However, OmpA and B and Fragment X are identifiable using patient sera (Panel B). FIG. 6 illustrates the specificity of the recombinant Fragment Y by western blot analysis. In FIG. 6 (a), no reactivity was observed against OmpA, OmpB or Fragment Y using control sera. However, OmpA and B and Fragment Y are clearly identifiable using patient sera (Panel B). FIG. 7 illustrates the specificity of the recombinant Fragment Z by western blot analysis.

Example 2

Use of OmpA Fragments X, Y and Z as Diagnostic Reagent

Assays using the recombinantly produced proteins include antibody-based assays such as enzyme-linked immunosorbent assays. As an illustration, the following procedure is provided, comprising the following steps: 1. Recombinant proteins represented by SEQ ID No. 3, 5 or 11 are immobilized, such as in 96-well plates. Alternatively, for increased sensitivity and specificity of the assay, both of the recombinant proteins represented by SEQ ID No. 3, 5 or 11 can be included together or immobilized separately but used in the same assay; 2. Wash off unreacted/unbound antigen. A preferred embodiment of the inventive method is to wash at least 3 times with wash buffer containing 0.1% polysorbate surfactant such as polyoxyethylene (20) sorbitan monolaurate; 3. Block unreacted sites. In a preferred embodiment, blocking of unreacted sites is accomplished with 5% skim milk in wash buffer).times.45 minutes and then rinsed three times. 4. React test sera to the bound antigen; 5. Plates are washed three times with wash buffer; 6. After incubating the test sera, the bound antibody-antigen is exposed to a probe. In a preferred embodiment, the probe is enzyme-labeled (e.g. peroxidase) anti-human immunoglobulin; 7. detecting bound probe. Detection of bound probe can be by any number of methods. In a preferred embodiment, detection is by measurement of enzymatic reaction of added substrate.

The above specific procedural outline is provided to illustrate the general method of using the fragments for the detection spotted fever group of Rickettsia infection, such as a R. Rickettisii infection. However, other iterations of the general procedure can be contemplated. Furthermore, a standard curve can be constructed by conducting antibody-base the above ELISA procedures with the recombinant proteins but utilizing a range of concentrations of specific antibody to R. rickettisii. The extent of measured binding of patient serum antibody is compared to a graphic representation of the binding of the R. rickettisii-specific antibody concentrations.

A prototype test using OmpA-X and OmpA-Y was build and its result is shown in FIG. 8.

Example 3

Prophetic Use of Recombinant R. Rickettisii OmpA Fragments X and Y as a Vaccine Component

The recombinantly produced polypeptides, because of their immunoreactivity to antibody in patient sera are excellent vaccine candidates. Accordingly, all or a fragment of the R. rickettisii proteins: Fragment X, Fragment Y or Fragment Z (SEQ ID No. 3, 5 or 11 respectively), or their respective DNA sequences (SEQ ID No. 4, 6, 12 respectively) incorporated into a suitable expression vector system, can be utilized as vaccine components. The method for induction of R. rickettisii immunity contains the following steps: a. administering an immunogenic composition in a unit dose range of 50 .mu.g to 2 mg, said immunogenic composition contains the entire or an immunogenic fragment of OmpA fragments X, Y or Z their amino acid sequences are set forth in SEQ ID No. 3, 5 or 11 respectively; b. administration of boosting dose of said immunogenic composition at least 1 week after priming dose with unit dose range of 50 .mu.g to 2 mg in a buffered aqueous solution, wherein an immune response is elicited.

An alternative method of immunizing is to administer DNA sequences encoding Fragments X, Y, or combinations thereof, inserted into a suitable expression system capable of expressing the fragments in vivo. Suitable expression systems can include viral expression vectors as well as a number of available DNA vector systems.

REFERENCE

1. Kelly D J, Richards A L, Temenak J, Strickman S, Dasch G A. The past and present threat of rickettsial diseases to military medicine and international public health. Clinical Infectious Diseases 2002; 34(Suppl 4):S145-69. 2. Walker D H. Rocky Mountain spotted fever: A disease in need of microbiological concern. Clinical Microbioloy Reviews 1989; 2(3):227-240. 3. Diaz-Montero C M, Feng H M, Crocquet-Valdes P A, Walker D H. Identification of protective components of two major outer membrane proteins of spotted fever group rickettsiae. American Journal of Medical Tropical Hygiene 2001; 65(4):371-378. 4. Croquet-Valdes P A, Diaz-Montero C M, Feng H M, Li H, Barrett A D T, Walker D H. Immunization with a portion of rickettsial outer membrane protein A stimulates protective immunity against spotted fever rickettsioses. Vaccine 2002; 20:979-988. 5. John W. Sumner, Kim G. Sims, Dana C. Jones and Burt E. Anderson. Protection of guinea-pigs from experimental Rocky Mountain spotted fever by immunization with baculovirus-expressed Rickettsia rickettsii rOmpA protein. Vaccine 1995; 13:29-35. 6. Vishwanath S, McDonald G A, Watkins N G. A recombinant Rickettsia conorii vaccine protects guinea pigs from experimental boutonneuse fever and Rocky Mountain spotted fever. Infect Immun 1990. 58:646-653. 7. R L Anacker, R E Mann, and C Gonzales. Reactivity of monoclonal antibodies to Rickettsia rickettsii with spotted fever and typhus group rickettsiae. J. Clin. Microbiol. 1987 25: 167-171. 8. R L Anacker, G A McDonald, R H List, and R E Mann. Neutralizing activity of monoclonal antibodies to heat-sensitive and heat-resistant epitopes of Rickettsia rickettsii surface proteins. Infect. Immun. 1987 55: 825-827.

SEQUENCE LISTINGS

1

2212249PRTRickettsia rickettsiiMISC_FEATUREOmpA Open Reading Frame 1Met Ala Asn Ile Ser Pro Lys Leu Phe Lys Lys Ala Ile Gln Gln Gly1 5 10 15Leu Lys Ala Ala Leu Phe Thr Thr Ser Thr Ala Ala Ile Met Leu Ser 20 25 30Ser Ser Gly Ala Leu Gly Val Ala Thr Gly Val Ile Ala Thr Asn Asn 35 40 45Asn Ala Ala Phe Ser Asn Asn Val Gly Asn Asn Asn Trp Asn Glu Ile 50 55 60Thr Ala Ala Gly Val Ala Asn Gly Thr Pro Ala Gly Gly Pro Gln Asn65 70 75 80Asn Trp Ala Phe Thr Tyr Gly Gly Asp Tyr Thr Val Thr Ala Asp Ala 85 90 95Ala Asp Arg Ile Ile Lys Ala Ile Asn Val Ala Gly Thr Thr Pro Val 100 105 110Gly Leu Asn Ile Thr Gln Asn Thr Val Val Gly Ser Ile Ile Thr Lys 115 120 125Gly Asn Leu Leu Pro Val Thr Leu Asn Ala Gly Lys Ser Leu Thr Leu 130 135 140Asn Gly Asn Asn Ala Val Ala Ala Asn His Gly Phe Asp Ala Pro Ala145 150 155 160Asp Asn Tyr Thr Gly Leu Gly Asn Ile Ala Leu Gly Gly Ala Asn Ala 165 170 175Ala Leu Ile Ile Gln Ser Ala Ala Pro Ser Lys Ile Thr Leu Ala Gly 180 185 190Asn Ile Asp Gly Gly Gly Ile Ile Thr Val Lys Thr Asp Ala Ala Ile 195 200 205Asn Gly Thr Ile Gly Asn Thr Asn Ala Leu Ala Thr Val Asn Val Gly 210 215 220Ala Gly Thr Ala Thr Leu Gly Gly Ala Val Ile Lys Ala Thr Thr Thr225 230 235 240Lys Leu Thr Asn Ala Ala Ser Val Leu Thr Leu Thr Asn Ala Asn Ala 245 250 255Val Leu Thr Gly Ala Ile Asp Asn Thr Thr Gly Gly Asp Asn Val Gly 260 265 270Val Leu Asn Leu Asn Gly Ala Leu Ser Gln Val Thr Gly Asp Ile Gly 275 280 285Asn Thr Asn Ser Leu Ala Thr Ile Ser Val Gly Ala Gly Thr Ala Thr 290 295 300Leu Gly Gly Ala Val Ile Lys Ala Thr Thr Thr Lys Leu Thr Asp Ala305 310 315 320Ala Ser Ala Val Lys Phe Thr Asn Pro Val Val Val Thr Gly Ala Ile 325 330 335Asp Asn Thr Gly Asn Ala Asn Asn Gly Ile Val Thr Phe Thr Gly Asn 340 345 350Ser Thr Val Thr Gly Asn Val Gly Asn Thr Asn Ala Leu Ala Thr Val 355 360 365Asn Val Gly Ala Gly Leu Leu Gln Val Gln Gly Gly Val Val Lys Ala 370 375 380Asn Thr Ile Asn Leu Thr Asp Asn Ala Ser Ala Val Thr Phe Thr Asn385 390 395 400Pro Val Val Val Thr Gly Ala Ile Asp Asn Thr Gly Asn Ala Asn Asn 405 410 415Gly Ile Val Thr Phe Thr Gly Asn Ser Thr Val Thr Gly Asp Ile Gly 420 425 430Asn Thr Asn Ala Leu Ala Thr Val Asn Val Gly Ala Gly Thr Ala Thr 435 440 445Leu Gly Gly Ala Val Ile Lys Ala Thr Thr Thr Lys Leu Thr Asn Ala 450 455 460Ala Ser Val Leu Thr Leu Thr Asn Ala Asn Ala Val Leu Thr Gly Ala465 470 475 480Ile Asp Asn Thr Thr Gly Gly Asp Asn Val Gly Val Leu Asn Leu Asn 485 490 495Gly Ala Leu Ser Gln Val Thr Gly Asn Ile Gly Asn Thr Asn Ser Leu 500 505 510Ala Thr Ile Ser Val Gly Ala Gly Thr Ala Thr Leu Gly Gly Ala Val 515 520 525Ile Lys Ala Thr Thr Thr Lys Leu Thr Asp Ala Ala Ser Ala Val Lys 530 535 540Phe Thr Asn Pro Val Val Val Thr Gly Ala Ile Asp Asn Thr Gly Asn545 550 555 560Ala Asn Asn Gly Ile Val Thr Phe Thr Gly Asn Ser Thr Val Thr Gly 565 570 575Asp Ile Gly Asn Thr Asn Ser Leu Ala Thr Ile Ser Val Gly Ala Gly 580 585 590Thr Ala Thr Leu Gly Gly Ala Val Ile Lys Ala Thr Thr Thr Lys Leu 595 600 605Thr Asn Ala Ala Ser Val Leu Thr Leu Thr Asn Ala Asn Ala Val Leu 610 615 620Thr Gly Ala Ile Asp Asn Thr Thr Gly Gly Asp Asn Val Gly Val Leu625 630 635 640Asn Leu Asn Gly Ala Leu Ser Gln Val Thr Gly Asp Ile Gly Asn Thr 645 650 655Asn Ser Leu Ala Thr Ile Ser Val Gly Ala Gly Thr Ala Thr Leu Gly 660 665 670Gly Ala Val Ile Lys Ala Thr Thr Thr Lys Ile Thr Asn Ala Val Ser 675 680 685Ala Val Lys Phe Thr Asn Pro Val Val Val Thr Gly Ala Ile Asp Ser 690 695 700Thr Gly Asn Ala Asn Asn Gly Ile Val Thr Phe Thr Gly Asn Ser Thr705 710 715 720Val Thr Gly Asp Ile Gly Asn Thr Asn Ala Leu Ala Thr Val Asn Val 725 730 735Gly Ala Gly Thr Ala Thr Leu Gly Gly Ala Val Ile Lys Ala Thr Thr 740 745 750Thr Lys Leu Thr Asn Ala Ala Ser Val Leu Thr Leu Thr Asn Ala Asn 755 760 765Ala Val Leu Thr Gly Ala Ile Asp Asn Thr Thr Gly Gly Asp Asn Val 770 775 780Gly Val Leu Asn Leu Asn Gly Ala Leu Ser Gln Val Thr Gly Asp Ile785 790 795 800Gly Asn Thr Asn Ser Leu Ala Thr Ile Ser Val Gly Ala Gly Thr Ala 805 810 815Thr Leu Gly Gly Ala Val Ile Lys Ala Thr Thr Thr Lys Leu Thr Asn 820 825 830Ala Ala Ser Val Leu Thr Leu Thr Asn Ala Asn Ala Val Leu Thr Gly 835 840 845Ala Val Asp Asn Thr Thr Gly Gly Asp Asn Val Gly Val Leu Asn Leu 850 855 860Asn Gly Ala Leu Ser Gln Val Thr Gly Asp Ile Gly Asn Thr Asn Ser865 870 875 880Leu Ala Thr Ile Ser Val Gly Ala Gly Thr Ala Thr Leu Gly Gly Ala 885 890 895Val Ile Lys Ala Thr Thr Thr Lys Leu Thr Asn Ala Ala Ser Val Leu 900 905 910Thr Leu Thr Asn Ala Asn Ala Val Leu Thr Gly Ala Ile Asp Asn Thr 915 920 925Thr Gly Gly Asp Asn Val Gly Val Leu Asn Leu Asn Gly Ala Leu Ser 930 935 940Gln Val Thr Gly Asp Ile Gly Asn Thr Asn Ser Leu Ala Thr Ile Ser945 950 955 960Val Gly Ala Gly Thr Ala Thr Leu Gly Gly Ala Val Ile Lys Ala Thr 965 970 975Thr Thr Lys Leu Thr Asp Ala Ala Ser Ala Val Lys Phe Thr Asn Pro 980 985 990Val Val Val Thr Gly Ala Ile Asp Asn Thr Gly Asn Ala Asn Asn Gly 995 1000 1005Ile Val Thr Phe Thr Gly Asn Ser Thr Val Thr Gly Asn Val Gly 1010 1015 1020Asn Thr Asn Ala Leu Ala Thr Val Asn Val Gly Ala Gly Leu Leu 1025 1030 1035Gln Val Gln Gly Gly Val Val Lys Ala Asn Thr Ile Asn Leu Thr 1040 1045 1050Asp Asn Ala Ser Ala Val Thr Phe Thr Asn Pro Val Val Val Thr 1055 1060 1065Gly Ala Ile Asp Asn Thr Gly Asn Ala Asn Asn Gly Ile Val Thr 1070 1075 1080Phe Thr Gly Asn Ser Thr Val Thr Gly Asn Val Gly Asn Thr Asn 1085 1090 1095Ala Leu Ala Thr Val Asn Val Gly Ala Gly Leu Leu Gln Val Gln 1100 1105 1110Gly Gly Val Val Lys Ala Asn Thr Ile Asn Leu Thr Asp Asn Ala 1115 1120 1125Ser Ala Val Thr Phe Thr Asn Pro Val Val Val Thr Gly Ala Ile 1130 1135 1140Asp Asn Thr Gly Asn Ala Asn Asn Gly Ile Val Thr Phe Thr Gly 1145 1150 1155Asn Ser Thr Val Thr Gly Asp Ile Gly Asn Thr Asn Ala Leu Ala 1160 1165 1170Thr Val Asn Val Gly Ala Gly Ile Thr Leu Gln Ala Gly Gly Ser 1175 1180 1185Leu Ala Ala Asn Asn Ile Asp Phe Gly Ala Arg Ser Thr Leu Glu 1190 1195 1200Phe Asn Gly Pro Leu Asp Gly Gly Gly Lys Ala Ile Pro Tyr Tyr 1205 1210 1215Phe Lys Gly Ala Ile Ala Asn Gly Asn Asn Ala Ile Leu Asn Val 1220 1225 1230Asn Thr Lys Leu Leu Thr Ala Ser His Leu Thr Ile Gly Thr Val 1235 1240 1245Ala Glu Ile Asn Ile Gly Ala Gly Asn Leu Phe Thr Ile Asp Ala 1250 1255 1260Ser Val Gly Asp Val Thr Ile Leu Asn Ala Gln Asn Ile Asn Phe 1265 1270 1275Arg Ala Arg Asp Ser Val Leu Val Leu Ser Asn Leu Thr Gly Val 1280 1285 1290Gly Val Asn Asn Ile Leu Leu Ala Ala Asp Leu Val Ala Pro Gly 1295 1300 1305Ala Asp Glu Gly Thr Val Val Phe Asn Gly Gly Val Asn Gly Leu 1310 1315 1320Asn Val Gly Ser Asn Val Ala Gly Thr Ala Arg Asn Ile Gly Asp 1325 1330 1335Gly Gly Gly Asn Lys Phe Asn Thr Leu Leu Ile Tyr Asn Ala Val 1340 1345 1350Thr Ile Thr Asp Asp Val Asn Leu Glu Gly Ile Gln Asn Val Leu 1355 1360 1365Ile Asn Lys Asn Ala Asp Phe Thr Ser Ser Thr Ala Phe Asn Ala 1370 1375 1380Gly Ala Ile Gln Ile Asn Asp Ala Thr Tyr Thr Ile Asp Ala Asn 1385 1390 1395Asn Gly Asn Leu Asn Ile Pro Ala Gly Asn Ile Gln Phe Ala His 1400 1405 1410Ala Asp Ala Gln Leu Val Leu Gln Asn Ser Ser Gly Asn Asp Arg 1415 1420 1425Thr Ile Thr Leu Gly Ala Asn Ile Asp Pro Asp Asn Asp Asp Glu 1430 1435 1440Gly Ile Val Ile Leu Asn Ser Val Thr Ala Gly Lys Lys Leu Thr 1445 1450 1455Ile Ala Gly Gly Lys Thr Phe Gly Gly Ala His Lys Leu Gln Thr 1460 1465 1470Ile Leu Phe Lys Gly Ala Gly Asp Cys Ser Thr Ala Gly Thr Thr 1475 1480 1485Phe Asn Thr Thr Asn Ile Val Leu Asp Ile Thr Gly Gln Leu Glu 1490 1495 1500Leu Gly Ala Thr Thr Ala Asn Val Val Leu Phe Asn Asp Ala Val 1505 1510 1515Gln Leu Thr Gln Thr Gly Asn Ile Gly Gly Phe Leu Asp Phe Asn 1520 1525 1530Ala Lys Asn Gly Met Val Thr Leu Asn Asn Asn Val Asn Val Ala 1535 1540 1545Gly Ala Val Gln Asn Thr Gly Gly Thr Asn Asn Gly Thr Leu Ile 1550 1555 1560Val Leu Gly Ala Ser Asn Leu Asn Arg Val Asn Gly Ile Ala Met 1565 1570 1575Leu Lys Val Gly Ala Gly Asn Val Thr Ile Ala Lys Gly Gly Lys 1580 1585 1590Val Lys Ile Gly Glu Ile Gln Gly Thr Gly Thr Asn Thr Leu Thr 1595 1600 1605Leu Pro Ala His Phe Asn Leu Thr Gly Ser Ile Asn Lys Thr Gly 1610 1615 1620Gly Gln Ala Leu Lys Leu Asn Phe Met Asn Gly Gly Ser Val Ser 1625 1630 1635Gly Val Val Gly Thr Ala Ala Asn Ser Val Gly Asp Ile Thr Thr 1640 1645 1650Ala Gly Ala Thr Ser Phe Ala Ser Ser Val Asn Ala Lys Gly Thr 1655 1660 1665Ala Thr Leu Gly Gly Thr Thr Ser Phe Ala Asn Thr Phe Thr Asn 1670 1675 1680Thr Gly Ala Val Thr Leu Ala Lys Gly Ser Ile Thr Ser Phe Ala 1685 1690 1695Lys Asn Val Thr Ala Thr Ser Phe Val Ala Asn Ser Ala Thr Ile 1700 1705 1710Asn Phe Ser Asn Ser Leu Ala Phe Asn Ser Asn Ile Thr Gly Gly 1715 1720 1725Gly Thr Thr Leu Thr Leu Gly Ala Asn Gln Val Thr Tyr Thr Gly 1730 1735 1740Thr Gly Ser Phe Thr Asp Thr Leu Thr Leu Asn Thr Thr Phe Asp 1745 1750 1755Gly Ala Ala Lys Ser Gly Gly Asn Ile Leu Ile Lys Ser Gly Ser 1760 1765 1770Thr Leu Asp Leu Ser Gly Val Ser Thr Leu Ala Leu Val Val Thr 1775 1780 1785Ala Thr Asn Phe Asp Met Asn Asn Ile Ser Pro Asp Thr Lys Tyr 1790 1795 1800Thr Val Ile Ser Ala Glu Thr Ala Gly Gly Leu Lys Pro Thr Ser 1805 1810 1815Lys Glu Asn Val Lys Ile Thr Ile Asn Asn Asp Asn Arg Phe Val 1820 1825 1830Asp Phe Thr Phe Asp Ala Ser Thr Leu Thr Leu Phe Ala Glu Asp 1835 1840 1845Ile Ala Ala Asp Val Ile Asp Gly Asp Phe Ala Pro Gly Gly Pro 1850 1855 1860Leu Ala Asn Ile Pro Asn Ala Ala Asn Ile Lys Lys Ser Leu Glu 1865 1870 1875Leu Met Glu Asp Ala Pro Asn Gly Ser Asp Ala Arg Gln Ala Phe 1880 1885 1890Asn Asn Phe Gly Leu Met Thr Pro Leu Gln Glu Ala Asp Ala Thr 1895 1900 1905Thr His Leu Ile Gln Asp Val Val Lys Pro Ser Asp Thr Ile Ala 1910 1915 1920Ala Val Asn Asn Gln Val Val Ala Ser Asn Ile Ser Ser Asn Ile 1925 1930 1935Thr Ala Leu Asn Ala Arg Met Asp Lys Val Gln Ser Gly Asn Lys 1940 1945 1950Gly Pro Val Ser Ser Gly Asp Glu Asp Met Asp Ala Lys Phe Gly 1955 1960 1965Ala Trp Ile Ser Pro Phe Val Gly Asn Ala Thr Gln Lys Met Cys 1970 1975 1980Asn Ser Ile Ser Gly Tyr Lys Ser Asp Thr Thr Gly Gly Thr Ile 1985 1990 1995Gly Phe Asp Gly Phe Val Ser Asp Asp Leu Ala Leu Gly Leu Ala 2000 2005 2010Tyr Thr Arg Ala Asp Thr Asp Ile Lys Leu Lys Asn Asn Lys Thr 2015 2020 2025Gly Asp Lys Asn Lys Val Glu Ser Asn Ile Tyr Ser Leu Tyr Gly 2030 2035 2040Leu Tyr Asn Val Pro Tyr Glu Asn Leu Phe Val Glu Ala Ile Ala 2045 2050 2055Ser Tyr Ser Asp Asn Lys Ile Arg Ser Lys Ser Arg Arg Val Ile 2060 2065 2070Ala Thr Thr Leu Glu Thr Val Gly Tyr Gln Thr Ala Asn Gly Lys 2075 2080 2085Tyr Lys Ser Glu Ser Tyr Thr Gly Gln Leu Met Ala Gly Tyr Thr 2090 2095 2100Tyr Met Met Pro Glu Asn Ile Asn Leu Thr Pro Leu Ala Gly Leu 2105 2110 2115Arg Tyr Ser Thr Ile Lys Asp Lys Gly Tyr Lys Glu Thr Gly Thr 2120 2125 2130Thr Tyr Gln Asn Leu Thr Val Lys Gly Lys Asn Tyr Asn Thr Phe 2135 2140 2145Asp Gly Leu Leu Gly Ala Lys Val Ser Ser Asn Ile Asn Val Asn 2150 2155 2160Glu Ile Val Leu Thr Pro Glu Leu Tyr Ala Met Val Asp Tyr Ala 2165 2170 2175Phe Lys Asn Lys Val Ser Ala Ile Asp Ala Arg Leu Gln Gly Met 2180 2185 2190Thr Ala Pro Leu Pro Thr Asn Ser Phe Lys Gln Ser Lys Thr Ser 2195 2200 2205Phe Asp Val Gly Val Gly Val Thr Ala Lys His Lys Met Met Glu 2210 2215 2220Tyr Arg Ile Asn Tyr Asp Thr Asn Ile Gly Ser Lys Tyr Phe Ala 2225 2230 2235Gln Gln Gly Ser Val Lys Val Arg Val Asn Phe 2240 224526750DNARickettsia rickettsiimisc_featureOmpA nucleotide sequence 2atggcgaata tttctccaaa attatttaaa aaagcaatac aacaaggtct taaagccgct 60ttattcacca cctcaaccgc agcgataatg ctgagtagta gcggggcact cggtgttgct 120acaggtgtta ttgctactaa taataatgca gcatttagta ataatgttgg caataataat 180tggaatgaga taacggctgc aggggtagct aatggtactc ctgctggcgg tcctcaaaac 240aattgggcat ttacttacgg tggtgattat actgtcactg cagatgcagc cgatcgtatt 300attaaggcta taaatgttgc gggtactact cccgtaggtc taaatattac tcaaaatact 360gtcgttggtt cgattataac gaaaggtaac ttgttgcctg ttactcttaa tgccggcaaa 420agcttaactt taaatggtaa taatgctgtt gctgcaaatc atggttttga tgcgcctgcc 480gataattata caggtttagg aaatatagct ttagggggag cgaatgctgc actaattata 540caatctgcag ctccgtcaaa gataacactt gcaggaaata tagatggagg aggtataata 600actgtcaaga cagatgctgc cattaacgga acaataggta atacaaatgc attagcaaca 660gtgaatgtag gagcaggtac agccacgtta gggggagcgg ttattaaagc tactacgact 720aaattaacga atgctgcgtc ggtattaacc cttacaaatg caaatgcagt attaacaggt 780gcgattgata acaccacagg cggtgataat gtaggtgtct taaatttaaa tggtgcatta 840agtcaagtaa ctggggatat aggtaataca aattcattag ccacgataag tgtaggagca 900ggtacagcca cgttaggggg agcggttatt aaagctacta cgactaagtt gacagatgct 960gcgtcagcag tgaaatttac gaatcctgta gtggtgactg gagcgataga taataccggt 1020aatgcaaata atggtatagt aacgtttacc ggtaatagta cagtaactgg gaatgtaggt 1080aatacaaatg cattagcaac agtgaatgta ggagcaggtt tgctacaagt acaaggtgga

1140gtggtaaaag caaatacaat aaacttaacg gataatgcgt cagcagtgac atttacgaat 1200cctgtagtgg tgaccggagc gatagataat accggtaatg caaataatgg tatagtaacg 1260tttaccggta atagtacagt aactggggat ataggtaata caaatgcatt agcaacagtg 1320aatgtaggag caggtacagc cacgctaggg ggagcggtta ttaaagctac tacgactaaa 1380ttaacgaatg ctgcgtcggt attaaccctt acaaatgcaa atgcagtatt aacaggtgcg 1440attgataaca ccacaggcgg tgataatgta ggtgtcttaa atttaaatgg tgcattaagt 1500caagtaactg ggaatatagg taatacaaat tcattagcca cgataagtgt aggagcaggt 1560acagccacgt tagggggagc ggttattaaa gctactacga ctaagttgac agatgctgcg 1620tcagcagtga aatttacgaa tcctgtagtg gtgactggag cgatagataa taccggtaat 1680gcaaataatg gtatagtaac gtttaccggt aatagtacag taactgggga tataggtaat 1740acaaattcat tagccacgat aagtgtagga gcaggtacag ccacgttagg gggagcagtt 1800attaaagcta ctacgactaa attaacgaat gctgcgtcgg tattaaccct tacaaatgca 1860aatgcagtat taacaggtgc gattgataac accacaggcg gtgataatgt aggtgtctta 1920aatttaaatg gtgcattaag tcaagtaact ggggatatag gtaatacaaa ttcattagcc 1980acgataagtg taggagcagg tacagccacg ttagggggag cggttattaa agctactacg 2040actaaaataa cgaatgctgt gtcagcagtg aaatttacga atcctgtagt ggtgaccgga 2100gcgatagata gtaccggtaa tgccaataat ggtatagtaa cgtttaccgg taatagtaca 2160gtaactgggg atataggtaa tacaaatgca ttagcaacag tgaatgtagg agcaggtaca 2220gccacgctag ggggagcggt tattaaagct actacgacta agttaacgaa tgctgcgtcg 2280gtattaaccc ttacaaatgc aaatgcagta ttaacaggtg cgattgataa caccacaggc 2340ggtgataatg taggtgtctt aaatttaaat ggtgcgttaa gtcaagtaac tggggatata 2400ggtaatacaa attcattagc cacgataagt gtaggagcag gtacagccac gttaggggga 2460gcggttatta aagctactac gactaaatta acgaatgctg cgtcggtatt aacccttaca 2520aatgcaaatg cagtattaac aggtgcggtt gataacacca caggcggtga taatgtaggt 2580gtcttaaatt taaatggtgc gttaagtcaa gtaactgggg atataggtaa tacaaattca 2640ttagccacga taagtgtagg agcaggtaca gccacgttag ggggagcggt tattaaagct 2700actacgacta aattaacgaa tgctgcgtcg gtattaaccc ttacaaatgc aaatgcagta 2760ttaacaggtg cgattgataa caccacaggc ggtgataatg taggtgtctt aaatttaaat 2820ggtgcgttaa gtcaagtaac tggggatata ggtaatacaa attcattagc cacgataagt 2880gtaggagcag gtacagccac gttaggggga gcggttatta aagctactac gactaagttg 2940acagatgctg cgtcagcagt gaaatttacg aatcctgtag tggtgaccgg agcgatagat 3000aataccggta atgcaaataa tggtatagta acgtttaccg gtaatagtac agtaactggg 3060aatgtaggta atacaaatgc attagcaaca gtgaatgtag gagcaggttt gctacaagta 3120caaggtggag tggtaaaagc aaatacaata aacttaacgg ataatgcgtc agcagtgaca 3180tttacgaatc ctgtagtggt gaccggagcg atagataata ccggtaatgc aaataatggt 3240atagtaacgt ttaccggtaa tagtacagta actgggaatg taggtaatac aaatgcatta 3300gcaacagtga atgtaggagc aggtttgcta caagtacaag gtggagtggt aaaagcaaat 3360acaataaact taacggataa tgcgtcagca gtgacattta cgaatcctgt agtggtgacc 3420ggagcgatag ataataccgg taatgcaaat aatggtatag taacgtttac cggtaatagt 3480acagtaactg gggatatagg taatacaaat gcattagcaa cagtgaatgt aggagcagga 3540ataacattac aagctggagg aagcctagct gcgaataata tagattttgg agccaggagt 3600actttagagt ttaacggacc tcttgatggt ggtggtaaag caatccctta ttattttaaa 3660ggagctatag caaacggcaa taatgctata ttaaatgtta atacaaagtt acttacggca 3720tctcatttaa ctataggaac agttgcagaa atcaatattg gagctggtaa tctttttaca 3780attgatgcaa gtgttggtga tgttactata ttaaatgctc aaaatattaa ttttagagct 3840cgagattctg ttttagtact ttctaactta accggagtcg gagtaaataa tatattatta 3900gcagctgatt tagtagctcc cggtgctgat gaaggtacgg tagtctttaa tggtggggtt 3960aatggcctga atgttgggag taatgtagca ggtaccgcta gaaatatcgg tgatggaggc 4020ggtaataaat ttaacacttt acttatttat aatgctgtta caataactga cgatgtaaat 4080ttagaaggta tacagaacgt gcttattaac aagaatgcag attttactag tagtacagca 4140tttaatgctg gtgctataca aataaacgat gctacttata cgattgatgc aaataatggt 4200aatttaaata taccggcagg aaatattcaa tttgcacatg cggatgctca attagtatta 4260caaaatagtt caggaaacga ccgtacgata acactaggtg cgaatataga ccctgataat 4320gacgatgagg gtatagtaat attaaattct gtaactgcag gaaaaaaatt aacgatagcc 4380ggaggcaaga cgtttggtgg agctcataag ttacaaacta tattgttcaa aggagcggga 4440gattgtagca cggcaggtac cacttttaat acaacaaata tagtacttga tattacaggt 4500caattagaac ttggagctac tacggcaaat gtagttttat ttaatgatgc tgttcaatta 4560actcaaaccg gtaatattgg cggtttctta gattttaatg caaaaaacgg tatggtaaca 4620ttaaataaca atgtaaatgt tgcgggagca gtccaaaata ccggcggtac taataacggt 4680acgttaatag ttttaggtgc aagtaatctt aatagagtaa acgggattgc tatgttaaaa 4740gtaggtgcag gaaatgtaac tattgccaaa ggcggtaaag ttaaaatcgg cgaaatccaa 4800ggtacaggca caaatacttt aacattacct gcacacttta acttaacagg cagcataaat 4860aaaaccggtg gtcaggctct gaagctaaac ttcatgaatg gcggtagtgt tagcggtgtt 4920gtagggactg cggctaattc ggttggtgat atcacaacgg caggtgctac aagttttgca 4980agcagtgtta acgcaaaagg tacggcgaca cttggcggta ctacaagttt tgccaataca 5040ttcactaata caggtgcggt tactttagcc aaaggttcta tcactagttt tgctaaaaat 5100gtaacggcta ccagctttgt agctaacagt gctactatta atttcagcaa tagcctagcc 5160tttaatagta atataacagg tggcggtact acacttactt taggtgcaaa tcaagtaaca 5220tatactggca ccggtagctt taccgatacg ctaaccttaa atactacttt tgacggagca 5280gctaagtcag gtggtaatat cttaattaaa tcaggtagta ctcttgattt atcaggggtt 5340tcaactttag cacttgttgt tactgctact aatttcgaca tgaataatat aagcccagat 5400acaaaatata cggtaatatc tgcagaaaca gcaggtggtt taaagcctac ttctaaagag 5460aatgttaaaa taactattaa taatgacaac cgttttgttg actttacttt tgatgcatcg 5520actttaacgt tatttgcaga agatatagct gcagatgtta tagatggaga ttttgcaccg 5580ggtggaccgc ttgcaaatat cccaaatgct gcaaatataa agaaatcgct tgagttaatg 5640gaggatgctc ccaatggttc agatgcacgt caagctttca ataactttgg tctaatgaca 5700ccgctacagg aagcagatgc tacaactcat ctcattcaag atgttgtaaa acctagcgac 5760actatagctg ccgttaataa tcaagttgta gcgagtaata tatcaagtaa tataactgct 5820ctaaatgcta gaatggataa agtacaatca gggaataaag gtcctgtttc ttctggtgat 5880gaagatatgg atgctaagtt tggtgcgtgg ataagcccgt ttgtcggtaa tgcaacgcag 5940aagatgtgta acagtataag tggttataag tctgatacaa ctggtggcac tataggtttt 6000gacggcttcg ttagcgatga tctagcactc ggacttgcat atacaagagc cgatactgac 6060attaagctaa aaaataataa aacgggcgat aagaataagg tagagagcaa catctattct 6120ttatacggtt tatataatgt accttatgaa aatctcttcg ttgaagctat agcatcttac 6180tcagataata agataagaag caaatcaaga cgtgttattg caacgacact agagactgtc 6240ggttatcaaa ctgcaaacgg taagtataaa tccgaaagct atacaggtca gttaatggct 6300ggttatacct atatgatgcc tgagaacatt aacttaacac cgctagctgg gcttagatat 6360tcgactatca aagataaggg ctataaggaa accggtacta cttaccaaaa tcttaccgtt 6420aaaggcaaga actataatac tttcgacggt ttactcggtg ctaaagtatc aagtaatatc 6480aatgtcaatg aaatagtgct aacacctgag ctttacgcaa tggtcgatta tgcattcaag 6540aataaagttt cggcgattga tgcaaggtta caaggtatga ctgctcctct tccaaccaac 6600agctttaagc aaagcaaaac aagttttgat gtcggtgtcg gtgttactgc taagcataaa 6660atgatggaat acaggattaa ctacgatacc aatatcggaa gtaagtattt cgctcagcaa 6720ggtagtgtaa aagttcgtgt taatttttaa 67503242PRTRickettsia rickettsiiMISC_FEATUREOmpA fragment X polypeptide 3Arg Asp Ser Val Leu Val Leu Ser Asn Leu Thr Gly Val Gly Val Asn1 5 10 15Asn Ile Leu Leu Ala Ala Asp Leu Val Ala Pro Gly Ala Asp Glu Gly 20 25 30Thr Val Val Phe Asn Gly Gly Val Asn Gly Leu Asn Val Gly Ser Asn 35 40 45Val Ala Gly Thr Ala Arg Asn Ile Gly Asp Gly Gly Gly Asn Lys Phe 50 55 60Asn Thr Leu Leu Ile Tyr Asn Ala Val Thr Ile Thr Asp Asp Val Asn65 70 75 80Leu Glu Gly Ile Gln Asn Val Leu Ile Asn Lys Asn Ala Asp Phe Thr 85 90 95Ser Ser Thr Ala Phe Asn Ala Gly Ala Ile Gln Ile Asn Asp Ala Thr 100 105 110Tyr Thr Ile Asp Ala Asn Asn Gly Asn Leu Asn Ile Pro Ala Gly Asn 115 120 125Ile Gln Phe Ala His Ala Asp Ala Gln Leu Val Leu Gln Asn Ser Ser 130 135 140Gly Asn Asp Arg Thr Ile Thr Leu Gly Ala Asn Ile Asp Pro Asp Asn145 150 155 160Asp Asp Glu Gly Ile Val Ile Leu Asn Ser Val Thr Ala Gly Lys Lys 165 170 175Leu Thr Ile Ala Gly Gly Lys Thr Phe Gly Gly Ala His Lys Leu Gln 180 185 190Thr Ile Leu Phe Lys Gly Ala Gly Asp Cys Ser Thr Ala Gly Thr Thr 195 200 205Phe Asn Thr Thr Asn Ile Val Leu Asp Ile Thr Gly Gln Leu Glu Leu 210 215 220Gly Ala Thr Thr Ala Asn Val Val Leu Phe Asn Asp Ala Val Gln Leu225 230 235 240Thr Gln4726DNARickettsia rickettsiimisc_featureOmpA Fragment X Nucleotide 4cgagattctg ttttagtact ttctaactta accggagtcg gagtaaataa tatattatta 60gcagctgatt tagtagctcc cggtgctgat gaaggtacgg tagtctttaa tggtggggtt 120aatggcctga atgttgggag taatgtagca ggtaccgcta gaaatatcgg tgatggaggc 180ggtaataaat ttaacacttt acttatttat aatgctgtta caataactga cgatgtaaat 240ttagaaggta tacagaacgt gcttattaac aagaatgcag attttactag tagtacagca 300tttaatgctg gtgctataca aataaacgat gctacttata cgattgatgc aaataatggt 360aatttaaata taccggcagg aaatattcaa tttgcacatg cggatgctca attagtatta 420caaaatagtt caggaaacga ccgtacgata acactaggtg cgaatataga ccctgataat 480gacgatgagg gtatagtaat attaaattct gtaactgcag gaaaaaaatt aacgatagcc 540ggaggcaaga cgtttggtgg agctcataag ttacaaacta tattgttcaa aggagcggga 600gattgtagca cggcaggtac cacttttaat acaacaaata tagtacttga tattacaggt 660caattagaac ttggagctac tacggcaaat gtagttttat ttaatgatgc tgttcaatta 720actcaa 7265250PRTRickettsia rickettsiiMISC_FEATUREOmpA Fragment Y polypeptide 5Val Thr Ala Thr Ser Phe Val Ala Asn Ser Ala Thr Ile Asn Phe Ser1 5 10 15Asn Ser Leu Ala Phe Asn Ser Asn Ile Thr Gly Gly Gly Thr Thr Leu 20 25 30Thr Leu Gly Ala Asn Gln Val Thr Tyr Thr Gly Thr Gly Ser Phe Thr 35 40 45Asp Thr Leu Thr Leu Asn Thr Thr Phe Asp Gly Ala Ala Lys Ser Gly 50 55 60Gly Asn Ile Leu Ile Lys Ser Gly Ser Thr Leu Asp Leu Ser Gly Val65 70 75 80Ser Thr Leu Ala Leu Val Val Thr Ala Thr Asn Phe Asp Met Asn Asn 85 90 95Ile Ser Pro Asp Thr Lys Tyr Thr Val Ile Ser Ala Glu Thr Ala Gly 100 105 110Gly Leu Lys Pro Thr Ser Lys Glu Asn Val Lys Ile Thr Ile Asn Asn 115 120 125Asp Asn Arg Phe Val Asp Phe Thr Phe Asp Ala Ser Thr Leu Thr Leu 130 135 140Phe Ala Glu Asp Ile Ala Ala Asp Val Ile Asp Gly Asp Phe Ala Pro145 150 155 160Gly Gly Pro Leu Ala Asn Ile Pro Asn Ala Ala Asn Ile Lys Lys Ser 165 170 175Leu Glu Leu Met Glu Asp Ala Pro Asn Gly Ser Asp Ala Arg Gln Ala 180 185 190Phe Asn Asn Phe Gly Leu Met Thr Pro Leu Gln Glu Ala Asp Ala Thr 195 200 205Thr His Leu Ile Gln Asp Val Val Lys Pro Ser Asp Thr Ile Ala Ala 210 215 220Val Asn Asn Gln Val Val Ala Ser Asn Ile Ser Ser Asn Ile Thr Ala225 230 235 240Leu Asn Ala Arg Met Asp Lys Val Gln Ser 245 2506750DNARickettsia rickettsiimisc_featureOmpA Fragment Y Nucleotide 6gtaacggcta ccagctttgt agctaacagt gctactatta atttcagcaa tagcctagcc 60tttaatagta atataacagg tggcggtact acacttactt taggtgcaaa tcaagtaaca 120tatactggca ccggtagctt taccgatacg ctaaccttaa atactacttt tgacggagca 180gctaagtcag gtggtaatat cttaattaaa tcaggtagta ctcttgattt atcaggggtt 240tcaactttag cacttgttgt tactgctact aatttcgaca tgaataatat aagcccagat 300acaaaatata cggtaatatc tgcagaaaca gcaggtggtt taaagcctac ttctaaagag 360aatgttaaaa taactattaa taatgacaac cgttttgttg actttacttt tgatgcatcg 420actttaacgt tatttgcaga agatatagct gcagatgtta tagatggaga ttttgcaccg 480ggtggaccgc ttgcaaatat cccaaatgct gcaaatataa agaaatcgct tgagttaatg 540gaggatgctc ccaatggttc agatgcacgt caagctttca ataactttgg tctaatgaca 600ccgctacagg aagcagatgc tacaactcat ctcattcaag atgttgtaaa acctagcgac 660actatagctg ccgttaataa tcaagttgta gcgagtaata tatcaagtaa tataactgct 720ctaaatgcta gaatggataa agtacaatca 750736DNARickettsia rickettsii 7ggtggtcata tgcgagattc tgttttagta ctttct 36837DNARickettsia rickettsii 8ggtggtctcg agttgagtta attgaacagc atcatta 37934DNARickettsia rickettsii 9ggtggtcata tggtaacggc taccagcttt gtag 341036DNARickettsia rickettsii 10ggtggtctcg agtgattgta ctttatccat tctagc 3611670PRTRickettsia rickettsiiMISC_FEATUREOmAp_Z polypeptide 11Arg Asp Ser Val Leu Val Leu Ser Asn Leu Thr Gly Val Gly Val Asn1 5 10 15Asn Ile Leu Leu Ala Ala Asp Leu Val Ala Pro Gly Ala Asp Glu Gly 20 25 30Thr Val Val Phe Asn Gly Gly Val Asn Gly Leu Asn Val Gly Ser Asn 35 40 45Val Ala Gly Thr Ala Arg Asn Ile Gly Asp Gly Gly Gly Asn Lys Phe 50 55 60Asn Thr Leu Leu Ile Tyr Asn Ala Val Thr Ile Thr Asp Asp Val Asn65 70 75 80Leu Glu Gly Ile Gln Asn Val Leu Ile Asn Lys Asn Ala Asp Phe Thr 85 90 95Ser Ser Thr Ala Phe Asn Ala Gly Ala Ile Gln Ile Asn Asp Ala Thr 100 105 110Tyr Thr Ile Asp Ala Asn Asn Gly Asn Leu Asn Ile Pro Ala Gly Asn 115 120 125Ile Gln Phe Ala His Ala Asp Ala Gln Leu Val Leu Gln Asn Ser Ser 130 135 140Gly Asn Asp Arg Thr Ile Thr Leu Gly Ala Asn Ile Asp Pro Asp Asn145 150 155 160Asp Asp Glu Gly Ile Val Ile Leu Asn Ser Val Thr Ala Gly Lys Lys 165 170 175Leu Thr Ile Ala Gly Gly Lys Thr Phe Gly Gly Ala His Lys Leu Gln 180 185 190Thr Ile Leu Phe Lys Gly Ala Gly Asp Cys Ser Thr Ala Gly Thr Thr 195 200 205Phe Asn Thr Thr Asn Ile Val Leu Asp Ile Thr Gly Gln Leu Glu Leu 210 215 220Gly Ala Thr Thr Ala Asn Val Val Leu Phe Asn Asp Ala Val Gln Leu225 230 235 240Thr Gln Thr Gly Asn Ile Gly Gly Phe Leu Asp Phe Asn Ala Lys Asn 245 250 255Gly Met Val Thr Leu Asn Asn Asn Val Asn Val Ala Gly Ala Val Gln 260 265 270Asn Thr Gly Gly Thr Asn Asn Gly Thr Leu Ile Val Leu Gly Ala Ser 275 280 285Asn Leu Asn Arg Val Asn Gly Ile Ala Met Leu Lys Val Gly Ala Gly 290 295 300Asn Val Thr Ile Ala Lys Gly Gly Lys Val Lys Ile Gly Glu Ile Gln305 310 315 320Gly Thr Gly Thr Asn Thr Leu Thr Leu Pro Ala His Phe Asn Leu Thr 325 330 335Gly Ser Ile Asn Lys Thr Gly Gly Gln Ala Leu Lys Leu Asn Phe Met 340 345 350Asn Gly Gly Ser Val Ser Gly Val Val Gly Thr Ala Ala Asn Ser Val 355 360 365Gly Asp Ile Thr Thr Ala Gly Ala Thr Ser Phe Ala Ser Ser Val Asn 370 375 380Ala Lys Gly Thr Ala Thr Leu Gly Gly Thr Thr Ser Phe Ala Asn Thr385 390 395 400Phe Thr Asn Thr Gly Ala Val Thr Leu Ala Lys Gly Ser Ile Thr Ser 405 410 415Phe Ala Lys Asn Val Thr Ala Thr Ser Phe Val Ala Asn Ser Ala Thr 420 425 430Ile Asn Phe Ser Asn Ser Leu Ala Phe Asn Ser Asn Ile Thr Gly Gly 435 440 445Gly Thr Thr Leu Thr Leu Gly Ala Asn Gln Val Thr Tyr Thr Gly Thr 450 455 460Gly Ser Phe Thr Asp Thr Leu Thr Leu Asn Thr Thr Phe Asp Gly Ala465 470 475 480Ala Lys Ser Gly Gly Asn Ile Leu Ile Lys Ser Gly Ser Thr Leu Asp 485 490 495Leu Ser Gly Val Ser Thr Leu Ala Leu Val Val Thr Ala Thr Asn Phe 500 505 510Asp Met Asn Asn Ile Ser Pro Asp Thr Lys Tyr Thr Val Ile Ser Ala 515 520 525Glu Thr Ala Gly Gly Leu Lys Pro Thr Ser Lys Glu Asn Val Lys Ile 530 535 540Thr Ile Asn Asn Asp Asn Arg Phe Val Asp Phe Thr Phe Asp Ala Ser545 550 555 560Thr Leu Thr Leu Phe Ala Glu Asp Ile Ala Ala Asp Val Ile Asp Gly 565 570 575Asp Phe Ala Pro Gly Gly Pro Leu Ala Asn Ile Pro Asn Ala Ala Asn 580 585 590Ile Lys Lys Ser Leu Glu Leu Met Glu Asp Ala Pro Asn Gly Ser Asp 595 600 605Ala Arg Gln Ala Phe Asn Asn Phe Gly Leu Met Thr Pro Leu Gln Glu 610 615 620Ala Asp Ala Thr Thr His Leu Ile Gln Asp Val Val Lys Pro Ser Asp625 630 635 640Thr Ile Ala Ala Val Asn Asn Gln Val Val Ala Ser Asn Ile Ser Ser 645 650 655Asn Ile Thr Ala Leu Asn Ala Arg Met Asp Lys Val Gln Ser 660 665 670122010DNARickettsia rickettsiimisc_featureOmpA_Z nucleotide 12cgagattctg ttttagtact ttctaactta accggagtcg gagtaaataa tatattatta 60gcagctgatt tagtagctcc

cggtgctgat gaaggtacgg tagtctttaa tggtggggtt 120aatggcctga atgttgggag taatgtagca ggtaccgcta gaaatatcgg tgatggaggc 180ggtaataaat ttaacacttt acttatttat aatgctgtta caataactga cgatgtaaat 240ttagaaggta tacagaacgt gcttattaac aagaatgcag attttactag tagtacagca 300tttaatgctg gtgctataca aataaacgat gctacttata cgattgatgc aaataatggt 360aatttaaata taccggcagg aaatattcaa tttgcacatg cggatgctca attagtatta 420caaaatagtt caggaaacga ccgtacgata acactaggtg cgaatataga ccctgataat 480gacgatgagg gtatagtaat attaaattct gtaactgcag gaaaaaaatt aacgatagcc 540ggaggcaaga cgtttggtgg agctcataag ttacaaacta tattgttcaa aggagcggga 600gattgtagca cggcaggtac cacttttaat acaacaaata tagtacttga tattacaggt 660caattagaac ttggagctac tacggcaaat gtagttttat ttaatgatgc tgttcaatta 720actcaaaccg gtaatattgg cggtttctta gattttaatg caaaaaacgg tatggtaaca 780ttaaataaca atgtaaatgt tgcgggagca gtccaaaata ccggcggtac taataacggt 840acgttaatag ttttaggtgc aagtaatctt aatagagtaa acgggattgc tatgttaaaa 900gtaggtgcag gaaatgtaac tattgccaaa ggcggtaaag ttaaaatcgg cgaaatccaa 960ggtacaggca caaatacttt aacattacct gcacacttta acttaacagg cagcataaat 1020aaaaccggtg gtcaggctct gaagctaaac ttcatgaatg gcggtagtgt tagcggtgtt 1080gtagggactg cggctaattc ggttggtgat atcacaacgg caggtgctac aagttttgca 1140agcagtgtta acgcaaaagg tacggcgaca cttggcggta ctacaagttt tgccaataca 1200ttcactaata caggtgcggt tactttagcc aaaggttcta tcactagttt tgctaaaaat 1260gtaacggcta ccagctttgt agctaacagt gctactatta atttcagcaa tagcctagcc 1320tttaatagta atataacagg tggcggtact acacttactt taggtgcaaa tcaagtaaca 1380tatactggca ccggtagctt taccgatacg ctaaccttaa atactacttt tgacggagca 1440gctaagtcag gtggtaatat cttaattaaa tcaggtagta ctcttgattt atcaggggtt 1500tcaactttag cacttgttgt tactgctact aatttcgaca tgaataatat aagcccagat 1560acaaaatata cggtaatatc tgcagaaaca gcaggtggtt taaagcctac ttctaaagag 1620aatgttaaaa taactattaa taatgacaac cgttttgttg actttacttt tgatgcatcg 1680actttaacgt tatttgcaga agatatagct gcagatgtta tagatggaga ttttgcaccg 1740ggtggaccgc ttgcaaatat cccaaatgct gcaaatataa agaaatcgct tgagttaatg 1800gaggatgctc ccaatggttc agatgcacgt caagctttca ataactttgg tctaatgaca 1860ccgctacagg aagcagatgc tacaactcat ctcattcaag atgttgtaaa acctagcgac 1920actatagctg ccgttaataa tcaagttgta gcgagtaata tatcaagtaa tataactgct 1980ctaaatgcta gaatggataa agtacaatca 201013242PRTRickettsia slovaca 13Leu Asp Ser Ala Leu Val Leu Ser Asn Leu Thr Gly Val Gly Val Asn1 5 10 15Asn Ile Leu Leu Ala Ala Asp Leu Val Ala Pro Gly Ala Asp Glu Gly 20 25 30Thr Val Val Phe Asn Gly Gly Val Asn Gly Leu Asn Ile Gly Ser Asn 35 40 45Val Ala Gly Thr Ala Arg Asn Ile Gly Asp Gly Gly Gly Asn Lys Phe 50 55 60Asn Thr Leu Leu Ile Tyr Asn Ala Val Thr Ile Thr Asp Asp Val Asn65 70 75 80Leu Glu Gly Ile Gln Asn Val Leu Ile Asn Asn Asn Ala Asp Phe Thr 85 90 95Ser Ser Thr Ala Phe Asn Ala Gly Ala Ile Gln Ile Asn Asp Ala Thr 100 105 110Tyr Thr Ile Asp Ala Asn Asn Gly Asn Leu Asn Ile Pro Ala Gly Asn 115 120 125Ile Gln Phe Ala His Ala Asp Ala Gln Leu Ile Leu Gln Asn Ser Ser 130 135 140Gly Asn Asp Arg Thr Ile Thr Leu Gly Ala Asn Ile Asp Pro Asp Asn145 150 155 160Asp Asp Glu Gly Ile Val Ile Leu Asn Ser Val Thr Ala Gly Lys Lys 165 170 175Leu Thr Ile Ala Gly Gly Lys Thr Phe Gly Gly Ala His Lys Leu Gln 180 185 190Thr Ile Val Phe Lys Gly Ala Gly Asp Cys Gly Ala Ala Gly Thr Thr 195 200 205Phe Asn Thr Thr Asn Ile Glu Leu Asn Ile Thr Gly Gln Leu Glu Leu 210 215 220Gly Ala Thr Thr Ala Asn Val Val Leu Phe Asn Asp Ala Val Gln Leu225 230 235 240Thr Gln14242PRTRickettsia honei 14Leu Asp Ser Ala Leu Val Leu Ser Asn Leu Thr Gly Val Gly Val Asn1 5 10 15Asn Ile Leu Leu Ala Ala Asp Leu Val Ala Pro Gly Ala Asp Glu Gly 20 25 30Thr Val Val Phe Asn Gly Gly Val Asn Gly Leu Asn Ile Gly Ser Asn 35 40 45Val Ala Gly Thr Ala Arg Asn Ile Gly Asp Gly Gly Gly Asn Lys Phe 50 55 60Asn Thr Leu Ser Ile Tyr Asn Ala Val Thr Ile Thr Asp Asp Val Asn65 70 75 80Leu Glu Gly Ile Gln Asn Val Leu Ile Asn Asp Asn Ala Asp Phe Thr 85 90 95Ser Ser Thr Ala Phe Asn Ala Gly Thr Ile Gln Ile Lys Asp Ala Thr 100 105 110Tyr Thr Ile Asp Ala Asn Asn Gly Asn Leu Asn Ile Pro Ala Gly Asn 115 120 125Ile Gln Phe Ala His Ala Asp Ala Gln Leu Ile Leu Gln Asn Ser Ser 130 135 140Gly Asn Asp Arg Thr Ile Thr Leu Gly Ala Asn Ile Asp Pro Asp Asn145 150 155 160Asp Asp Glu Gly Ile Val Ile Leu Asn Ser Val Thr Ala Gly Lys Lys 165 170 175Leu Thr Ile Ala Gly Gly Lys Met Phe Gly Gly Ala His Lys Leu Gln 180 185 190Thr Ile Val Phe Lys Gly Ala Gly Asn Cys Gly Ala Ala Gly Thr Thr 195 200 205Phe Asn Thr Thr Asn Ile Val Leu Asp Ile Thr Gly Gln Leu Glu Leu 210 215 220Gly Ala Thr Thr Ala Ser Val Val Leu Phe Asn Asp Ala Val Gln Leu225 230 235 240Thr Gln15242PRTRickettsia rickettsii 15Arg Asp Ser Val Leu Val Leu Ser Asn Leu Thr Gly Val Gly Val Asn1 5 10 15Asn Ile Leu Leu Ala Ala Asp Leu Val Ala Pro Gly Ala Asp Glu Gly 20 25 30Thr Val Val Phe Asn Gly Gly Val Asn Gly Leu Asn Val Gly Ser Asn 35 40 45Val Ala Gly Thr Ala Arg Asn Ile Gly Asp Gly Gly Gly Asn Lys Phe 50 55 60Asn Thr Leu Leu Ile Tyr Asn Ala Val Thr Ile Thr Asp Asp Val Asn65 70 75 80Leu Glu Gly Ile Gln Asn Val Leu Ile Asn Lys Asn Ala Asp Phe Thr 85 90 95Ser Ser Thr Ala Phe Asn Ala Gly Ala Ile Gln Ile Asn Asp Ala Thr 100 105 110Tyr Thr Ile Asp Ala Asn Asn Gly Asn Leu Asn Ile Pro Ala Gly Asn 115 120 125Ile Gln Phe Ala His Ala Asp Ala Gln Leu Val Leu Gln Asn Ser Ser 130 135 140Gly Asn Asp Arg Thr Ile Thr Leu Gly Ala Asn Ile Asp Pro Asp Asn145 150 155 160Asp Asp Glu Gly Ile Val Ile Leu Asn Ser Val Thr Ala Gly Lys Lys 165 170 175Leu Thr Ile Ala Gly Gly Lys Thr Phe Gly Gly Ala His Lys Leu Gln 180 185 190Thr Ile Leu Phe Lys Gly Ala Gly Asp Cys Ser Thr Ala Gly Thr Thr 195 200 205Phe Asn Thr Thr Asn Ile Val Leu Asp Ile Thr Gly Gln Leu Glu Leu 210 215 220Gly Ala Thr Thr Ala Asn Val Val Leu Phe Asn Asp Ala Val Gln Leu225 230 235 240Thr Gln16242PRTRickettsia peacockii 16Leu Asp Ser Val Leu Val Leu Ser Asn Leu Thr Gly Val Gly Val Asn1 5 10 15Asn Ile Leu Leu Ala Ala Asp Leu Val Ala Pro Gly Ala Asp Glu Gly 20 25 30Thr Ile Val Phe Asn Gly Gly Val Asn Gly Leu Asn Ile Gly Ser Asn 35 40 45Val Ala Gly Thr Ala Arg Asn Ile Gly Asp Gly Gly Gly Asn Lys Phe 50 55 60Asn Thr Leu Leu Ile Tyr Asn Ala Val Thr Ile Thr Asp Asp Val Asn65 70 75 80Leu Glu Gly Ile Gln Asn Val Leu Ile Asn Asn Asn Ala Asp Phe Thr 85 90 95Ser Ser Thr Ala Phe Asn Ala Gly Ala Ile Gln Ile Asn Asp Ala Thr 100 105 110Tyr Thr Ile Asp Ala Asn Asn Gly Asn Leu Asn Ile Pro Ala Gly Asn 115 120 125Ile Gln Phe Ala His Ala Asp Ala Gln Leu Val Leu Gln Asn Ser Ser 130 135 140Gly Asn Asp Arg Thr Ile Thr Leu Gly Ala Asn Ile Asp Pro Asp Asn145 150 155 160Asp Asp Glu Gly Ile Val Ile Leu Asn Ser Val Thr Ala Gly Lys Lys 165 170 175Leu Thr Ile Ala Gly Gly Lys Thr Phe Gly Gly Ala His Lys Leu Gln 180 185 190Thr Ile Leu Phe Lys Gly Ala Gly Asp Cys Ser Ala Ala Gly Thr Thr 195 200 205Phe Asn Thr Thr Asn Ile Val Leu Asp Ile Thr Gly Gln Leu Glu Leu 210 215 220Gly Ala Thr Thr Ala Asn Val Val Leu Phe Asn Asp Ala Val Gln Leu225 230 235 240Thr Gln17242PRTRickettsia parkeri 17Leu Asp Ser Gly Leu Val Leu Ser Asn Leu Thr Gly Val Gly Val Asn1 5 10 15Asn Ile Leu Leu Ala Ala Asp Leu Val Ala Pro Gly Ala Asp Glu Gly 20 25 30Thr Val Ile Phe Asn Gly Gly Val Asn Gly Leu Asn Ile Gly Ser Asn 35 40 45Val Ala Gly Thr Ala Arg Asn Ile Gly Asp Gly Gly Gly Asn Lys Phe 50 55 60Asn Thr Leu Leu Ile Asp Asn Ala Val Thr Ile Thr Asp Asp Val Asn65 70 75 80Leu Glu Gly Ile Gln Asn Val Leu Ile Asn Asn Lys Ala Asp Phe Thr 85 90 95Ser Ser Thr Ala Phe Asn Ala Gly Ala Ile Gln Ile Asn Asp Ala Thr 100 105 110Tyr Thr Ile Asp Ala Asn Asn Gly Asn Leu Asn Ile Pro Ala Gly Asn 115 120 125Ile Gln Phe Ala His Ala Asp Ala Gln Leu Ile Leu Gln Asn Ser Ser 130 135 140Gly Asn Asp Arg Thr Ile Thr Leu Gly Ala Asn Ile Asp Pro Asp Asn145 150 155 160Asp Asp Glu Gly Ile Val Ile Leu Asn Ser Val Thr Ala Gly Lys Lys 165 170 175Leu Thr Ile Ala Gly Gly Lys Thr Phe Gly Gly Ala His Lys Leu Gln 180 185 190Thr Ile Val Phe Lys Gly Ala Gly Asp Cys Gly Thr Ala Gly Thr Thr 195 200 205Phe Asn Thr Thr Asn Ile Val Leu Asp Ile Thr Gly Gln Leu Glu Leu 210 215 220Gly Ala Thr Thr Ala Asn Val Val Leu Phe Lys Asp Ala Val Gln Leu225 230 235 240Thr Gln18250PRTRickettsia honei 18Val Thr Ala Thr Ser Phe Val Ala Asn Ser Ala Thr Ile Asn Phe Gly1 5 10 15Asn Ser Leu Ala Phe Asn Ser Asn Ile Thr Gly Ser Gly Thr Thr Leu 20 25 30Thr Leu Gly Ala Asn Gln Val Thr Tyr Thr Gly Thr Gly Ser Phe Thr 35 40 45Asp Thr Leu Thr Leu Asn Thr Thr Phe Asp Gly Ala Ala Lys Ser Gly 50 55 60Gly Asn Ile Leu Ile Lys Ser Gly Ser Thr Leu Asp Leu Ser Gly Val65 70 75 80Ser Thr Leu Ala Leu Val Val Thr Ala Thr Asn Phe Asp Met Asn Asn 85 90 95Ile Ser Pro Asp Thr Lys Tyr Thr Val Ile Ser Ala Glu Thr Ala Gly 100 105 110Gly Leu Lys Pro Thr Pro Lys Glu Asn Val Lys Ile Thr Ile Asn Asn 115 120 125Asp Asn Arg Phe Val Asp Phe Thr Phe Asp Ala Ser Thr Leu Thr Leu 130 135 140Phe Ala Glu Asp Ile Ala Ala Asp Val Ile Asp Glu Asp Phe Ala Pro145 150 155 160Gly Gly Pro Leu Ala Asn Ile Pro Asn Ala Ala Asn Ile Lys Lys Ser 165 170 175Leu Glu Leu Met Glu Asp Ala Pro Asn Gly Ser Asp Ala Arg Gln Ala 180 185 190Phe Asn Asn Phe Gly Leu Met Thr Pro Leu Gln Glu Ala Asp Ala Thr 195 200 205Thr His Leu Met Gln Asp Val Val Lys Pro Ser Asp Thr Ile Ala Ala 210 215 220Val Asn Asn Gln Val Val Ala Ser Asn Ile Ser Ser Asn Ile Thr Ala225 230 235 240Leu Asn Ala Arg Met Asp Lys Val Gln Ala 245 25019250PRTRickettsia conorii 19Val Thr Ala Thr Ser Phe Val Ala Asn Ser Ala Thr Ile Asn Phe Gly1 5 10 15Asn Ser Leu Ala Phe Asn Ser Asn Ile Thr Gly Ser Gly Thr Thr Leu 20 25 30Thr Leu Gly Ala Asn Gln Val Thr Tyr Thr Gly Thr Gly Ser Phe Thr 35 40 45Asp Thr Leu Thr Leu Asn Thr Thr Phe Asp Gly Ala Ala Lys Ser Gly 50 55 60Gly Asn Ile Leu Ile Lys Ser Gly Ser Thr Leu Asp Leu Ser Gly Val65 70 75 80Ser Asn Leu Ala Leu Val Val Thr Ala Thr Asn Phe Asp Met Asn Asn 85 90 95Ile Ser Pro Asp Thr Lys Tyr Thr Val Ile Ser Ala Glu Thr Ala Gly 100 105 110Gly Leu Lys Pro Thr Pro Lys Glu Asn Val Lys Ile Thr Ile Asn Asn 115 120 125Asp Asn Arg Phe Val Asp Phe Thr Phe Asp Ala Ser Thr Leu Thr Leu 130 135 140Phe Ala Glu Asp Ile Ala Ala Gly Val Ile Asp Glu Asp Phe Ala Pro145 150 155 160Gly Gly Pro Leu Ala Asn Ile Pro Asn Ala Ala Asn Ile Lys Lys Ser 165 170 175Leu Glu Leu Met Glu Asp Ala Pro Asn Gly Ser Asp Ala Arg Gln Ala 180 185 190Phe Asn Asn Phe Gly Leu Met Thr Pro Leu Gln Glu Ala Asp Ala Thr 195 200 205Thr His Leu Met Gln Asp Val Val Lys Pro Ser Asp Thr Ile Ala Ala 210 215 220Val Asn Asn Gln Val Val Ala Ser Asn Ile Ser Ser Asn Ile Thr Ala225 230 235 240Leu Asn Ala Arg Met Asp Lys Val Gln Ala 245 25020250PRTRickettsia slovaca 20Val Thr Ala Thr Ser Phe Val Ala Asn Ser Ala Thr Ile Asn Phe Gly1 5 10 15Asn Ser Leu Ala Phe Asn Ser Asn Ile Thr Gly Ser Gly Thr Thr Leu 20 25 30Thr Leu Gly Ala Asn Gln Val Thr Tyr Thr Gly Thr Gly Ser Phe Thr 35 40 45Asp Thr Leu Thr Leu Asn Thr Thr Phe Asp Gly Ala Ala Lys Ser Gly 50 55 60Gly Asn Ile Leu Ile Lys Ser Gly Ser Thr Leu Asp Leu Ser Gly Val65 70 75 80Ser Thr Leu Ala Leu Val Val Thr Ala Thr Asn Phe Asp Met Asn Asn 85 90 95Ile Ser Pro Asp Thr Lys Tyr Thr Val Ile Ser Ala Glu Thr Ala Gly 100 105 110Gly Leu Lys Pro Thr Pro Lys Glu Asn Val Lys Ile Thr Ile Asn Asn 115 120 125Asp Asn Arg Phe Val Asp Phe Thr Phe Asp Ala Ser Thr Leu Thr Leu 130 135 140Phe Ala Glu Asp Ile Ala Ala Asp Val Ile Asp Glu Asp Phe Ala Pro145 150 155 160Gly Gly Pro Leu Ala Asn Ile Pro Asn Ala Ala Asn Ile Lys Lys Ser 165 170 175Leu Glu Leu Met Ala Asp Ala Pro Asn Gly Ser Asp Ala Arg Gln Ala 180 185 190Phe Asn Asn Phe Gly Leu Met Thr Pro Leu Gln Glu Ala Asp Ala Thr 195 200 205Thr His Leu Met Gln Asp Val Val Lys Pro Ser Asp Thr Ile Ala Ala 210 215 220Val Asn Asn Gln Val Val Ala Ser Asn Ile Ser Ser Asn Ile Thr Ala225 230 235 240Leu Asn Ala Arg Met Asp Lys Val Gln Ala 245 25021250PRTRickettsia parkeri 21Val Thr Ala Thr Ser Phe Val Ala Asn Ser Ala Thr Ile Asn Phe Gly1 5 10 15Asn Ser Leu Ala Phe Asn Ser Asn Ile Thr Gly Ser Gly Thr Thr Leu 20 25 30Thr Leu Gly Ala Asn Gln Val Thr Tyr Asn Gly Thr Gly Ser Phe Thr 35 40 45Asp Thr Leu Thr Leu Asn Thr Thr Phe Asp Gly Ala Ala Lys Ser Gly 50 55 60Gly Asn Ile Leu Ile Lys Ser Gly Ser Thr Leu Asp Leu Ser Gly Val65 70 75 80Ser Thr Leu Ala Leu Val Val Thr Ala Thr Asn Phe Asp Met Asn Asn 85 90 95Ile Ser Pro Asp Thr Lys Tyr Thr Val Ile Ser Ala Glu Thr Ala Gly 100 105 110Gly Leu Lys Pro Thr Pro Lys Glu Asn Val Lys Ile Thr Ile Asn Asn 115 120 125Asp Asn Arg Phe Val Asp Phe Thr Phe Asp Ala Ser Thr Leu Thr Leu 130 135 140Phe Ala Glu Asp Ile Ala Ala Asp Val Ile Asp Lys Asp Phe Ala Pro145 150 155 160Gly Gly Pro Leu Ala Asn

Ile Pro Asn Ala Ala Asn Ile Lys Lys Ser 165 170 175Leu Glu Leu Met Gly Asp Ala Pro Asn Gly Ser Asp Ala Arg Gln Ala 180 185 190Phe Asn Asn Phe Gly Leu Met Thr Pro Leu Gln Glu Ala Asp Ala Thr 195 200 205Thr His Leu Met Gln Asp Val Val Lys Pro Ser Asp Thr Ile Ala Ala 210 215 220Val Asn Asn Gln Val Val Ala Ser Asn Ile Ser Ser Asn Ile Thr Ala225 230 235 240Leu Asn Ala Arg Met Asp Lys Val Gln Val 245 25022250PRTRickettsia rickettsii 22Val Thr Ala Thr Ser Phe Val Ala Asn Ser Ala Thr Ile Asn Phe Ser1 5 10 15Asn Ser Leu Ala Phe Asn Ser Asn Ile Thr Gly Gly Gly Thr Thr Leu 20 25 30Thr Leu Gly Ala Asn Gln Val Thr Tyr Thr Gly Thr Gly Ser Phe Thr 35 40 45Asp Thr Leu Thr Leu Asn Thr Thr Phe Asp Gly Ala Ala Lys Ser Gly 50 55 60Gly Asn Ile Leu Ile Lys Ser Gly Ser Thr Leu Asp Leu Ser Gly Val65 70 75 80Ser Thr Leu Ala Leu Val Val Thr Ala Thr Asn Phe Asp Met Asn Asn 85 90 95Ile Ser Pro Asp Thr Lys Tyr Thr Val Ile Ser Ala Glu Thr Ala Gly 100 105 110Gly Leu Lys Pro Thr Ser Lys Glu Asn Val Lys Ile Thr Ile Asn Asn 115 120 125Asp Asn Arg Phe Val Asp Phe Thr Phe Asp Ala Ser Thr Leu Thr Leu 130 135 140Phe Ala Glu Asp Ile Ala Ala Asp Val Ile Asp Gly Asp Phe Ala Pro145 150 155 160Gly Gly Pro Leu Ala Asn Ile Pro Asn Ala Ala Asn Ile Lys Lys Ser 165 170 175Leu Glu Leu Met Glu Asp Ala Pro Asn Gly Ser Asp Ala Arg Gln Ala 180 185 190Phe Asn Asn Phe Gly Leu Met Thr Pro Leu Gln Glu Ala Asp Ala Thr 195 200 205Thr His Leu Ile Gln Asp Val Val Lys Pro Ser Asp Thr Ile Ala Ala 210 215 220Val Asn Asn Gln Val Val Ala Ser Asn Ile Ser Ser Asn Ile Thr Ala225 230 235 240Leu Asn Ala Arg Met Asp Lys Val Gln Ser 245 250