CN114957408B - PreG modified protein of rabies virus and application thereof - Google Patents

PreG modified protein of rabies virus and application thereof Download PDF

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CN114957408B
CN114957408B CN202110209081.6A CN202110209081A CN114957408B CN 114957408 B CN114957408 B CN 114957408B CN 202110209081 A CN202110209081 A CN 202110209081A CN 114957408 B CN114957408 B CN 114957408B
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CN114957408A (en
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王弋
李静怡
刘昕
郑飞
陈怡林
段巧梅
吴先戈
彭小珍
龙夏婷
邱福剑
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Guangzhou Yuanbo Pharmaceutical Technology Co ltd
Dongguan Bosheng Biological Technology Co ltd
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Dongguan Bosheng Biological Technology Co ltd
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Abstract

The invention belongs to the technical field of rabies virus recombinant proteins, and particularly relates to preG modified protein of rabies virus and application thereof. The preG modified protein is obtained by mutating, inserting or replacing amino acids of a qc connection region (Leu 257-Val 273) in rabies virus preG protein into other amino acids, wherein the other amino acids comprise proline. The amino acid sequence of preG modified protein is shown as sequence table SEQ ID NO. 2-SEQ ID NO. 35. The helix region of preG protein qC region is stabilized, the formation of redundant helix structure is prevented to cause the folding, and the unfolding and folding are prevented to form postG structure, so that the conformation of preG is stabilized, the stability of the structure is ensured, the protein structure expression quantity is improved, meanwhile, the immunogenicity is also improved, and the vaccine with large expression quantity and soluble expression is formed.

Description

PreG modified protein of rabies virus and application thereof
Technical Field
The invention belongs to the technical field of rabies virus recombinant proteins, and particularly relates to preG modified protein of rabies virus and application thereof.
Background
Rabies is a 100% fatal zoonotic disease that severely jeopardizes public health and life safety, caused by rabies virus (Rabies virus, RABV) of lyssavirus Lyssavirus in rhabdoviridae Rhabdovorodae. The pathogenic agent causing rabies in China is mainly RABV, the number of the RABV population types in China is 7 (China I-VI), the 7 th newly found population is from the cases of the Tibetan autonomous region in 2017, the types and the numbers of epidemic populations are different from each other, for example, the Yunnan province of the border of the ground and the Hunan province of the traditional high-rise region, and the number of populations is up to 4. At present, no effective treatment means exists, and the rabies vaccine inoculation is the only effective preventive measure.
The rabies vaccine in China goes through the stages of sheep brain, rabbit brain nerve tissue vaccine, cell culture concentrated vaccine, cell culture purified vaccine without adjuvant and the like. The rabies vaccine is only a local mouse kidney cell vaccine and a Vero cell purified vaccine for the people in China. The animal rabies live vaccine virus produced and marketed in China is introduced from abroad in 1979, and is suitable for the low-generation virus (LEP) of rabies chicken embryo of Flury strain grown on a BHK21 cell line. Rabies, canine distemper, parainfluenza, adenosis and parvovirus which are successfully developed in 2008 are put into the market. Since live vaccine viruses may have a recovery mutation in vivo to cause morbidity and have potential safety hazards, in recent years, inactivated vaccines are becoming important, and at present, canine vaccines in China mainly comprise live vaccines and inactivated vaccines, but rabies inactivated vaccines for animals are gradually replacing attenuated live vaccines.
The RABV genome encodes 5 structural proteins, nucleoprotein (N protein), phosphoprotein (P protein), matrix protein (M protein), transmembrane glycoprotein (G protein) and viral polymerase protein (L protein).
Apoptosis occurring after RABV infection is mainly caused by G protein, and the weaker the virulence of the virus, the more severe the apoptosis induced. The G protein is a trimeric transmembrane protein existing on the surface of a viral envelope, is the only glycoprotein on the surface of RV virus and is combined with a receptor on the surface of cells. The G protein is the only surface antigen for stimulating the organism to produce neutralizing antibodies, and can induce T cells and stimulate the organism to produce cellular immunity, so that the G protein can not only remove viruses in the Central Nervous System (CNS), but also induce nerve cells to undergo apoptosis. Plays a key role in rabies virus pathogenesis and immunity. So the rabies virus G protein has wide application in vaccine development, antibody detection and other aspects.
As viruses of the rhabdoviridae family, there are two conformations of the G proteins of VSV, RABV: since the RABV G protein of natural structure is preG under alkaline condition and postG under acidic condition, the structure is not stable. There are two structural states for rabies virus G protein structure, pre-fusion G protein (preG) with a curved hairpin structure on the viral surface and post-fusion G protein (postG) that mediates the membrane fusion extended conformation. The G protein on the viral surface exists mostly in the form of a bent hairpin structure (preG).
To establish infection, RABV will first bind to host receptors and then enter the cell via an endocytic pathway. The endocytic acid environment triggers the reverse rotation of the RABV-G partial region, resulting in a conformational change. As the pH decreases, it transitions from a pre-fusion state to a post-fusion state to mediate membrane fusion. It is thought that RABV-G is capable of reverting to its natural preG conformation upon transport through the acidic environment of the Golgi apparatus. This pH-dependent conformational change was also confirmed in the G protein of VSV virus of the same rhabdoviridae.
Since RV G protein of natural structure is preG conformation under alkaline condition and postG conformation under acidic condition, its structure is not stable. At the same time, the preG conformation changes gradually to postG conformation along with the storage time and stability, so that the antigenic site is reduced, and the immunogenicity is reduced. As a main antigen, the configuration of the antigen can influence the immunogenicity of the antigen, so that the preG structure of the antigen is stabilized, the immunogenicity of the antigen can be effectively improved, and the immunoprotection effect is enhanced. Thus stabilizing the pre-fusion state of viral membrane proteins is a hotspot in current vaccine research.
Disclosure of Invention
Aiming at the problems, the invention provides preG modified protein of rabies virus and application thereof. The amino acid of the connecting region of the protein qC of the mutant rabies virus preG is proline, and the helical structure of the Leu257-Val273 region of the protein preG of the rabies virus is destroyed, so that the protein structure expression quantity and the immunogenicity are improved.
The technical content of the invention is as follows:
The invention provides a preG modified protein of rabies virus, which is obtained by mutating, inserting or replacing amino acids of a qC connection region (a qC region and a qC-connected CD region spiral region (Leu 257-Val 273)) in a rabies virus preG protein into other amino acids; the helical structure of preG protein amino acid is destroyed, so that the conformation of preG is stabilized, and preG protein cannot be changed into an extended conformation from a folded hairpin conformation;
The other amino acids include proline.
The number of sites for the amino terminal mutation or insertion includes one or more.
The site of amino acid mutation or insertion comprises one of the amino acids of the qC junction region of preG protein and the helical region (Leu 257-Val 273) of the qC junction CD region.
The amino acid sequence of the rabies virus preG protein is shown in a sequence table SEQ ID NO. 1;
the amino acid sequence of preG protein after site mutation comprises the amino acid sequence shown as SEQ ID NO. 2-SEQ ID NO.18 of the sequence table;
the amino acid sequence of preG protein after site insertion comprises the amino acid sequence shown as sequence table SEQ ID NO. 19-SEQ ID NO. 35;
the invention also provides application of the preG modified protein of the rabies virus in vaccine development and antibody detection; the method is applied to the preparation of vaccines, inactivated vaccines, attenuated vaccines or viral vector vaccines.
The invention also provides a preparation method of the preG modified protein vaccine of rabies virus, which comprises the following steps:
1) Constructing a vaccine: synthesizing preG modified protein sequences onto an expression vector, constructing a recombinant expression plasmid, and then transforming and transfecting to obtain preG modified protein vaccine of the soluble rabies virus;
2) Purifying.
The expression vector in the step 1) comprises a prokaryotic expression vector and a eukaryotic expression vector;
The expression vector comprises an insect virus vector, a yeast expression vector, a CHO expression vector or other virus expression vectors.
The purification operation of the step 2) comprises pretreatment, EDTA and alkali resistant Ni filler affinity chromatography and ultrafiltration concentration dialysis treatment of culture supernatant after preG modified protein vaccine centrifugation;
The pretreatment comprises the steps of centrifuging a baculovirus insect system, adding imidazole into a culture supernatant to a final concentration of 5 mM-50 mM, regulating the conductance to 10 ms/cm-50 ms/cm by using NaCl, regulating the pH to 6.0-8.0, filtering by using a membrane below 0.45um, and purifying the filtrate by chromatography;
the operation of the affinity chromatography comprises the following steps:
1) The upstream construction designs a histidine tag at the C-terminal, so that high-purity target protein can be conveniently and quickly obtained, and the affinity chromatography filler is EDTA and alkali resistant Ni filler; the imidazole concentration, the conductivity and the pH value of the filler balance liquid are the same as those of the pretreatment liquid: 10 mM-0.1M PB (Na 2HPO4,NaH2PO4); naCl concentration is 50 mM-0.6M; imidazole concentration is 5-50 mM; the pH is 6.2-8.0; the column height is 5-25 cm, and the flow speed is 30-300 cm/h; the balance volume is 2 CV-20 CV;
2) Sampling the filtered sample, wherein the sampling flow rate is 30 cm/h-300 cm/h, and the sampling amount is 2 mg-10 mg of filler per mL;
3) After the sample is loaded, the column is washed by the balance liquid until the base line is flat, and the flow rate is 30cm/h to 300cm/h;
4) The target protein with histidine tag is bound on the column under the conditions, and part of the target protein is non-specifically adsorbed on the column, so that the target protein can be washed off under the conditions without being washed off under the conditions by selecting proper conditions, thereby ensuring the improvement of purity and the reduction of recovery rate;
The impurity washing buffer solution mainly comprises 10 mM-0.1M PB (Na 2HPO4,NaH2PO4), 50 mM-0.6M NaCl concentration, 10-80 mM imidazole concentration, pH of 6.2-8.0 and flow rate of 60 cm/h-300 cm/h;
Eluting, namely eluting target protein by using an elution buffer solution after eluting impurities, wherein the elution buffer solution comprises 10 mM-0.1M PB (Na 2HPO4,NaH2PO4), 20 mM-0.6M NaCl concentration and 100 mM-1M imidazole concentration, the pH value is 6.2-8.0, and the flow rate is 30 cm/h-300 cm/h;
The ultrafiltration concentration dialysis treatment comprises the following steps: because the affinity chromatography collection liquid has high-concentration imidazole, and the protein is not easy to store after no stable component is purified, buffer replacement is needed, an ultrafiltration method is selected for buffer replacement, the ultrafiltration membrane package size is 3.5 KD-30 KD, and ultrafiltration buffers include but are not limited to: 10 mM-0.1M PB (Na 2HPO4,NaH2PO4), 1-3% mannitol, 1-4% arginine hydrochloride, pH 6.0-8.0;
the process is mainly characterized by simple process route, high purity after purification, high recovery rate, purity after purification of more than 90 percent and recovery rate of more than 85 percent;
The ultrafiltered sample is vaccine stock solution, and the vaccine stock solution and the adjuvant are prepared into vaccine finished products to be final products.
The beneficial effects of the invention are as follows:
according to the preG modified protein of the rabies virus, amino acid of a qC connection region (Leu 257-Val 273) in the rabies virus preG protein is mutated, inserted or replaced by other amino acid, so that a spiral region of a qC region of the preG protein is stabilized, a redundant spiral structure is prevented from being formed to cause folding, the folding is prevented from being unfolded to form a postG structure, the conformation of the preG protein is stabilized, the structural stability of the protein is ensured, the protein structure expression quantity of the protein is improved, meanwhile, the immunogenicity of the protein is improved, and a vaccine with large expression quantity and soluble expression is formed;
The preG modified protein of rabies virus is used for preparing vaccines, and after the prepared vaccines are immunized, high-level neutralizing antibodies can be generated, free viruses can be effectively neutralized, and replication of the viruses is inhibited. The preG modified protein of the rabies virus has wide application in the aspects of rabies virus vaccine development, antibody detection and the like.
Drawings
FIG. 1 shows the PCR identification of the recombinant plasmids of group 6 of example 1;
FIG. 2 shows the result of PCR electrophoresis of 6 recombinant bacmid of example 1;
FIG. 3 shows the WB assay results of the P0 generation protein expression of example 1;
FIG. 4 shows the WB assay results of the culture supernatant protein expression in the reactor of example 1;
FIG. 5 shows the SDS-PAGE results of the protein expression of example 1;
FIG. 6 shows the WB assay results of the purified protein of example 1;
FIG. 7 shows the results of experiments on the binding capacities of mutant rabies G protein with preG and postG antibodies in different pH solutions of example 1;
FIG. 8 shows the results of IgG antibody level detection after immunization of the different rabies preG modified protein vaccines of example 1;
FIG. 9 shows the results of an antibody neutralization assay after immunization with the modified protein vaccine of the different rabies preG of example 1.
Detailed Description
The application is described in further detail below with reference to specific embodiments and the accompanying drawings, it being understood that these embodiments are only for the purpose of illustrating the application and not for the purpose of limiting the same, and that various modifications of the application, which are equivalent to those skilled in the art, will fall within the scope of the appended claims after reading the present application.
All materials and reagents of the invention are materials and reagents of the conventional market unless specified otherwise.
Examples
Preparation of preG modified protein of rabies virus and vaccine thereof:
1. Construction of recombinant plasmids
PreG proteins consist of the following domains: the method comprises the steps of inserting proline between any amino acid sites in a qC connection region (Leu 257-Val 273) of an outer membrane region (1-439 aa), a transmembrane region (440-461 aa) and an inner membrane region (462-550 aa), changing preG proteins from a folded hairpin conformation to an extended conformation, fusing the two conformations in a tandem manner, adopting a codon optimization system to design and synthesize the recombinant plasmid on a pFastBac1 vector, constructing preG modified protein recombinant plasmids with 5 qC connection regions (Leu 257-Val 273) and mutated into proline by any amino acid, and respectively mutating the proline into 260, 265, 268, 270 and 273 amino acids, and respectively numbering plasmids RV-preG-pFastbac-260、RV-preG-pFastbac-265、RV-preG-pFastbac-268、RV-preG-pFastbac-270、RV-preG-pFastbac-273. to simultaneously construct plasmids RV-preG-pFastbac of genes with preG modified proteins.
2. Construction of recombinant bacmid
Transforming the recombinant plasmid RV-preG-pFastbac、RV-preG-pFastbac-260、RV-preG-pFastbac-265、RV-preG-pFastbac-268、RV-preG-pFastbac-270、RV-preG-pFastbac-273 into E.coli DH10Bac competent cells, carrying out blue-white spot screening recombinants (shown in figure 2) on an LB plate containing X-preGal/IPTpreG, selecting 5 white colonies from the recombinants, carrying out PCR identification, wherein the size of a target fragment is 800bp, and the PCR identification result is shown in figure 1, wherein the 5 white single colonies all carry target genes, so that the plasmid expression is correct;
2 PCR identified positive recombinants are picked for amplification culture, the alkaline lysis method is used for separating the DNA of the stem grains, the result of the electrophoresis of the stem grains is shown in figure 2, the concentration of the stem grains is higher, and the band expression is correct.
3. Baculovirus-insect system expression of rabies virus preG modified protein subunit vaccine
Bacmid-carrier complex preparation (10 mL): preparing two sterile centrifuge tubes, adding 0.5mL Grace's culture medium and 30 μg sterile rod particles into one sterile centrifuge tube, mixing, adding 0.5mL Grace's culture medium and 90 μl EZ-trans transfection reagent into the other sterile centrifuge tube, and mixing; transferring all liquid in the centrifuge tube containing the transfection reagent into the centrifuge tube containing the rod particles, uniformly mixing, and standing for 15-20 minutes at room temperature to prepare a rod particle-carrier compound; taking out cells from the biochemical shaking table, adding the prepared rod particle-carrier compound while shaking, shaking uniformly, and placing the mixture back into the biochemical shaking table for culturing at 28 ℃ and 120 rpm.
Virus harvesting and expression verification: appropriate amount of SF9 cells in exponential phase were diluted with fresh SF9 medium to a density of 1X 10 6/mL and inoculated into 125mL shake flasks, 20mL. According to MOI=1-5, P0 virus infection is inoculated, 0.5mL is taken for a Western Blot experiment on days 4-5, and the result is shown in FIG. 3, which shows that RV-preG-pFastbac、RV-preG-pFastbac-260、RV-preG-pFastbac-265、RV-preG-pFastbac-268、RV-preG-pFastbac-270、RV-preG-pFastbac-273 has protein expression on SF9 cells, and the expression forms are mainly culture supernatants.
And harvesting viruses on the 4 th to 5 th days, and preserving at-80 ℃.
And (3) performing enlarged culture in a reactor: and diluting a proper amount of SF9 cells in an exponential phase with fresh SF9 culture medium to make the density of the SF9 cells be 1 multiplied by 10 6/mL, inoculating the SF9 cells into a 250mL shaking flask, inoculating 50mL of the SF9 cells to the P2 virus infection according to MOI=1-5, and carrying out the infection at 28 ℃ and 120rpm. Seed toxicity is determined after 3-4 days of culture: SF9 cells (density 1×10 6/mL) =5% -10% seeding expands to 500mL; seed toxicity is determined after 3-4 days of culture: SF9 cells (density 1×10 6/mL) =5% -10% were inoculated into a 7L reactor, culture volume was 5L, culture conditions pH 6.5.+ -. 0.1, DO 40%, 28 ℃, 100rpm. And (5) culturing for 5-7 days, and harvesting culture supernatant. The protein expression condition of the culture supernatant is detected by Western Blot experiments, and the result is shown in figure 4, which shows that the reactor expansion culture protein expression quantity is higher and can be used for the next purification experiment.
Baculovirus-insect system expression RVG-260p, RVG-265p, RVG-268p, RVG-270p, RVG-273p proteins were expressed by the above procedure.
4. Purification of modified protein subunit vaccine of rabies preG
Sample pretreatment
Taking 500mL of culture supernatant expressed by an insect rod-shaped system of 6 proteins, adding imidazole to a final concentration of 50mM, and fully stirring until the mixture is completely dissolved after the addition;
The above treatment solution was adjusted to pH 7.0 with NaCl to 40ms/cm, filtered with 0.45um membrane, and the filtrate was used for chromatographic purification.
(II) affinity chromatography
(1) The affinity chromatography filler is EDTA and alkali resistant Ni filler; equilibration solution, 20mM PB, naCl concentration 0.3M, imidazole concentration 10mM, pH7.2; the column height is 5cm, and the flow rate is 30cm/h; balance volume 10CV;
(2) Sampling the filtered sample, wherein the sampling flow rate is 30cm/h, and the sampling amount is 2 mg-10 mg of filler per mL;
(3) After the sample is loaded, the column is washed by a balance liquid until the base line is flat, and the flow rate is 30cm/h;
(4) Washing: wash buffer 20mM PB, naCl concentration 0.3M, imidazole concentration 30mM, pH7.2, flow 30cm/h;
(5) Eluting: the elution buffer was 20mM PB, naCl concentration 0.3M, imidazole concentration 300mM, pH7.2, flow rate 30cm/h.
(III) ultrafiltration dialysis
The affinity chromatography collection liquid has high concentration imidazole, the ultrafiltration method is adopted for buffer replacement, the ultrafiltration membrane bag size is 10KD, and the ultrafiltration buffer solution is adopted: 20mMPB (Na 2HPO4,NaH2PO4), mannitol 1.5%, arginine hydrochloride 2%, pH7.4, and dialysis multiple 6 volumes. Concentrating to a concentration of 2mg/mL after dialysis;
Purity after purification by coomassie brilliant blue staining analysis by the above method, purity of RVG-260p, RVG-265p, RVG-268p, RVG-270p, RVG-273p was 93.1%, 92.8%, 93.2%, 93%, 92.5%, 93.5% respectively. The recovery rates were calculated to be 90.5%,90.2%,91%,90.6%,89.9% and 90.8% by content detection.
Identification of purified proteins: taking 15 mu L of each purified protein, mixing with an equal amount of 2x electrophoresis sample adding buffer solution, carrying out SDS-PAGE electrophoresis, wherein the electrophoresis result is shown in figure 5, a part of the proteins are dyed by coomassie brilliant blue, the purification condition is observed, the molecular weight of a sample lane 3 in figure 5 is the target molecular weight, and the coomassie-dyed result has almost no other impurity bands; and the other part of the membrane is transferred to carry out Western blot identification, and the WB detection result is shown in figure 6, wherein the molecular weight corresponding to the sample band of the lane 3 is the target molecular weight.
5. Antibody binding Capacity experiments for different conformational preG proteins
Murine anti-preG and postG antibodies were diluted with coating buffer (pH 9.6) to a concentration of 0.2 μg/ml and coated overnight at 4 ℃. Then blocked with 5% skimmed milk in PBS for 1 hour at room temperature. After blocking, 50. Mu.l of 200. Mu.g/ml histidine-tagged RVG, RVG-260p, RVG-265p, RVG-268p, RVG-270p, RVG-273p proteins in phosphate buffer at pH5.5 and 8.0, respectively, were diluted 5-fold, added to the wells, incubated for one hour at 37℃and then diluted anti-His mouse antibody was added at a ratio of 1:3000 for 1.5h, followed by goat anti-mouse IgG-HRP incubation for 1h. The color development was added and reacted for 3 minutes in the dark, and after termination with 2M HCl, the absorbance was read at 450 nm.
The results show that under different pH values, RVG-260p, RVG-265p, RVG-268p, RVG-270p and RVG-273p proteins can be combined with postG protein antibodies, and the combination capacities are not very different; however, for the binding capacity of preG antibodies, several proteins differ little from the binding capacity of preG antibody at ph 8.0; however, at pH5.5, where RVG-268p, RVG-270p, RVG-273p binding capacity was significantly higher than the original RVG, RVG-260p, showing that RVG-268p, RVG-270p, RVG-273p mutations stabilized its preG conformation, the results are shown in FIG. 7.
Binding of the above proteins to preG and postG specific antibodies, respectively, showed that the mutated preG modified protein was able to bind to both antibodies. The mutant RVG-268p, RVG-270p and RVG-273p modified proteins have no significant decrease in their binding ability to preG antibodies at low pH.
6. Preparation of subunit vaccine
The W/O/W water-in-oil-in-water adjuvant is 201 adjuvant purchased from French SEPPIC company, has the effect of enhancing immunity, can better improve cellular immunity, is the best adjuvant technology in inactivated vaccine, has small anaphylactic reaction when water is injected at the outermost layer, has oil at the second layer, and has long protection time; the third layer is again water, again to reduce allergic reactions in the body. Adding 201 adjuvants into concentrated and purified rabies RVG, RVG-260p, RVG-265p, RVG-268p, RVG-270p and RVG-273p according to the ratio of 1:1, and mixing uniformly with protein content of 100 μg/ml.
7. Immunization of subunit vaccine
Mice were randomly divided into a PBS control group and six protein immunized groups of 16 each. The mice of each group are immunized by leg intramuscular injection, the immunization dose is 100 mu L/mouse (the protein content is 100 mu g/mL), the immunization time is 0, 3 and 7d, the blood is collected from the 7 th and 14 d th veins after the first needle immunization, the serum is separated, and the serum is preserved at the temperature of minus 80 ℃.
8. ELISA detection of IgG antibodies in immunized mice
Serum from 14 d mice after initial immunization was taken and assayed for IgG levels using ELISA methods, following the IgG ELISA kit instructions. The test data is shown in FIG. 8, with RVG-265p, RVG-268p, RVG-270p, RVG-273p vaccine groups each producing higher antibody levels, and RVG, RVG-260p vaccine groups having lower antibody levels.
9. Post-immunization antibody neutralization assay
Culturing rabies virus cell-adapted strain CVS-11 by using a BHK-21 cell line, measuring the working concentration of fluorescent antibody by using infected cells, and measuring half-number infection amount (TCID 50) of CVS-11 cells by using a fluorescent antibody staining method; the neutralizing antibody level was determined from the serum of 7 d and 14 d mice after the initial needle immunization. As shown in figure 9, the neutralization capacity of RVG-265p, RVG-268p, RVG-270p and RVG-273p vaccine group antibodies is obviously higher than that of RVG, RVG-260p and vaccine group antibodies.
10. Toxicity attack protection experiment
Mice were randomly assigned to PBS control and RVG-265p, RVG-268p, RVG-270p, RVG-273p immunized groups of 16 animals each. 0.1 mL/dose (protein content 10. Mu.g/dose) was intraperitoneally injected at each of 0, 7 d. Mice were pre-titrated with 50 LD50 CVS-11 virus, 0.03 mL each, intracranially at 14 d after the first immunization. The survival rate was calculated by continuously observing 14 d, recording the death, counting the number of mice dead after 5 th d and presenting with typical rabies. The mice of each experimental group were observed for abnormal conditions such as contracture, erectile wool, convulsion, and cramp, and for feeding, excretion, and mental performance. Immunization results: the PBS control group starts to show clinical symptoms at the 7 d th challenge, which are Mao Luan, contracture, extreme excitation and the like, and gradually dies in 8-12 d. The vaccine group mice have no abnormal body weight, no symptoms of rabies virus infection such as contracture, mania, mao Luan and the like, and all survive.
The preG modified protein can be applied to the aspects of equal vaccine development and antibody detection, and the method for preparing the vaccine is not limited to the aspects, but can also be used for preparing an inactivated vaccine, an attenuated vaccine or a viral vector vaccine, and the expression vector used for preparing the vaccine is not limited to the aspects, but also comprises a yeast expression vector, a CHO expression vector or a viral expression vector, and the preparation method and the effect are similar to the aspects, and are not repeated herein.
In conclusion, the preG modified protein vaccine of rabies virus can effectively neutralize free virus, inhibit the replication of the virus, form a vaccine which has large expression quantity and can be expressed in a soluble way, and improve the effective immunogenicity while improving the protein structure expression quantity.
Sequence listing
<110> Dongguan Bosheng biotechnology Co., guangzhou Yuan Bo medicine Co., ltd
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Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
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Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
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Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
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Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
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His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
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Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
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Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
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Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
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Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu His Leu Val
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Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu
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Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu
290 295 300
Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys
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Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn
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Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys His
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Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala
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Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
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Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
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Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
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His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
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Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
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Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
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Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
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Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
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Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
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Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
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Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
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Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
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Pro Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu His Leu Val
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Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys
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Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp
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Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His
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Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala
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Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe Val
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Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
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Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
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Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
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His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
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Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
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Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
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Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
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Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
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Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu
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His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
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Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
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Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
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Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
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Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
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Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
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Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
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Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
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His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
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Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
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Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
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Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
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Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
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Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
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Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
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Leu Val Asn Pro His Asp Phe Arg Ser Asp Glu Ile Glu His Leu Val
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Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu
290 295 300
Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys
305 310 315 320
Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn
325 330 335
Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys His
340 345 350
Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp
355 360 365
Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His
370 375 380
Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala
385 390 395 400
Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe Val
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Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp Leu
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Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His His
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His
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Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu Pro Asp Phe Arg Ser Asp Glu Ile Glu His Leu Val
260 265 270
Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu
275 280 285
Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu
290 295 300
Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys
305 310 315 320
Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn
325 330 335
Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys His
340 345 350
Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp
355 360 365
Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His
370 375 380
Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala
385 390 395 400
Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe Val
405 410 415
Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp Leu
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Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His His
435 440 445
His
<210> 7
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Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Pro Phe Arg Ser Asp Glu Ile Glu His Leu Val
260 265 270
Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu
275 280 285
Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu
290 295 300
Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys
305 310 315 320
Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn
325 330 335
Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys His
340 345 350
Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp
355 360 365
Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His
370 375 380
Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala
385 390 395 400
Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe Val
405 410 415
Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp Leu
420 425 430
Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His His
435 440 445
His
<210> 8
<211> 449
<212> PRT
<213> Artificial Sequence
<400> 8
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Pro Arg Ser Asp Glu Ile Glu His Leu Val
260 265 270
Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu
275 280 285
Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu
290 295 300
Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys
305 310 315 320
Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn
325 330 335
Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys His
340 345 350
Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp
355 360 365
Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His
370 375 380
Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala
385 390 395 400
Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe Val
405 410 415
Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp Leu
420 425 430
Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His His
435 440 445
His
<210> 9
<211> 449
<212> PRT
<213> Artificial Sequence
<400> 9
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Pro Ser Asp Glu Ile Glu His Leu Val
260 265 270
Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu
275 280 285
Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu
290 295 300
Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys
305 310 315 320
Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn
325 330 335
Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys His
340 345 350
Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp
355 360 365
Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His
370 375 380
Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala
385 390 395 400
Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe Val
405 410 415
Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp Leu
420 425 430
Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His His
435 440 445
His
<210> 10
<211> 449
<212> PRT
<213> Artificial Sequence
<400> 10
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Pro Asp Glu Ile Glu His Leu Val
260 265 270
Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu
275 280 285
Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu
290 295 300
Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys
305 310 315 320
Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn
325 330 335
Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys His
340 345 350
Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp
355 360 365
Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His
370 375 380
Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala
385 390 395 400
Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe Val
405 410 415
Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp Leu
420 425 430
Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His His
435 440 445
His
<210> 11
<211> 449
<212> PRT
<213> Artificial Sequence
<400> 11
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Ser Pro Glu Ile Glu His Leu Val
260 265 270
Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu
275 280 285
Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu
290 295 300
Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys
305 310 315 320
Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn
325 330 335
Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys His
340 345 350
Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp
355 360 365
Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His
370 375 380
Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala
385 390 395 400
Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe Val
405 410 415
Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp Leu
420 425 430
Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His His
435 440 445
His
<210> 12
<211> 449
<212> PRT
<213> Artificial Sequence
<400> 12
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Ser Asp Pro Ile Glu His Leu Val
260 265 270
Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu
275 280 285
Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu
290 295 300
Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys
305 310 315 320
Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn
325 330 335
Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys His
340 345 350
Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp
355 360 365
Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His
370 375 380
Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala
385 390 395 400
Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe Val
405 410 415
Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp Leu
420 425 430
Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His His
435 440 445
His
<210> 13
<211> 449
<212> PRT
<213> Artificial Sequence
<400> 13
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Pro Glu His Leu Val
260 265 270
Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu
275 280 285
Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu
290 295 300
Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys
305 310 315 320
Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn
325 330 335
Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys His
340 345 350
Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp
355 360 365
Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His
370 375 380
Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala
385 390 395 400
Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe Val
405 410 415
Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp Leu
420 425 430
Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His His
435 440 445
His
<210> 14
<211> 449
<212> PRT
<213> Artificial Sequence
<400> 14
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Pro His Leu Val
260 265 270
Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu
275 280 285
Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu
290 295 300
Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys
305 310 315 320
Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn
325 330 335
Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys His
340 345 350
Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp
355 360 365
Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His
370 375 380
Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala
385 390 395 400
Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe Val
405 410 415
Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp Leu
420 425 430
Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His His
435 440 445
His
<210> 15
<211> 449
<212> PRT
<213> Artificial Sequence
<400> 15
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu Pro Leu Val
260 265 270
Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu
275 280 285
Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu
290 295 300
Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys
305 310 315 320
Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn
325 330 335
Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys His
340 345 350
Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp
355 360 365
Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His
370 375 380
Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala
385 390 395 400
Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe Val
405 410 415
Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp Leu
420 425 430
Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His His
435 440 445
His
<210> 16
<211> 449
<212> PRT
<213> Artificial Sequence
<400> 16
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu His Pro Val
260 265 270
Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu
275 280 285
Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu
290 295 300
Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys
305 310 315 320
Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn
325 330 335
Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys His
340 345 350
Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp
355 360 365
Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His
370 375 380
Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala
385 390 395 400
Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe Val
405 410 415
Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp Leu
420 425 430
Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His His
435 440 445
His
<210> 17
<211> 449
<212> PRT
<213> Artificial Sequence
<400> 17
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu His Leu Pro
260 265 270
Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu
275 280 285
Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu
290 295 300
Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys
305 310 315 320
Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn
325 330 335
Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys His
340 345 350
Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp
355 360 365
Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His
370 375 380
Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala
385 390 395 400
Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe Val
405 410 415
Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp Leu
420 425 430
Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His His
435 440 445
His
<210> 18
<211> 449
<212> PRT
<213> Artificial Sequence
<400> 18
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu His Leu Val
260 265 270
Pro Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu
275 280 285
Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu
290 295 300
Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys
305 310 315 320
Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn
325 330 335
Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys His
340 345 350
Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp
355 360 365
Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His
370 375 380
Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala
385 390 395 400
Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe Val
405 410 415
Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp Leu
420 425 430
Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His His
435 440 445
His
<210> 19
<211> 450
<212> PRT
<213> Artificial Sequence
<400> 19
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Pro Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu His Leu
260 265 270
Val Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu
275 280 285
Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His
290 295 300
Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn
305 310 315 320
Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp
325 330 335
Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys
340 345 350
His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro
355 360 365
Asp Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln
370 375 380
His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu
385 390 395 400
Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe
405 410 415
Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp
420 425 430
Leu Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His
435 440 445
His His
450
<210> 20
<211> 450
<212> PRT
<213> Artificial Sequence
<400> 20
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Pro Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu His Leu
260 265 270
Val Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu
275 280 285
Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His
290 295 300
Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn
305 310 315 320
Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp
325 330 335
Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys
340 345 350
His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro
355 360 365
Asp Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln
370 375 380
His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu
385 390 395 400
Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe
405 410 415
Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp
420 425 430
Leu Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His
435 440 445
His His
450
<210> 21
<211> 450
<212> PRT
<213> Artificial Sequence
<400> 21
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Pro Leu His Asp Phe Arg Ser Asp Glu Ile Glu His Leu
260 265 270
Val Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu
275 280 285
Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His
290 295 300
Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn
305 310 315 320
Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp
325 330 335
Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys
340 345 350
His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro
355 360 365
Asp Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln
370 375 380
His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu
385 390 395 400
Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe
405 410 415
Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp
420 425 430
Leu Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His
435 440 445
His His
450
<210> 22
<211> 450
<212> PRT
<213> Artificial Sequence
<400> 22
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu Pro His Asp Phe Arg Ser Asp Glu Ile Glu His Leu
260 265 270
Val Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu
275 280 285
Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His
290 295 300
Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn
305 310 315 320
Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp
325 330 335
Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys
340 345 350
His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro
355 360 365
Asp Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln
370 375 380
His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu
385 390 395 400
Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe
405 410 415
Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp
420 425 430
Leu Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His
435 440 445
His His
450
<210> 23
<211> 450
<212> PRT
<213> Artificial Sequence
<400> 23
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Pro Asp Phe Arg Ser Asp Glu Ile Glu His Leu
260 265 270
Val Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu
275 280 285
Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His
290 295 300
Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn
305 310 315 320
Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp
325 330 335
Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys
340 345 350
His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro
355 360 365
Asp Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln
370 375 380
His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu
385 390 395 400
Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe
405 410 415
Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp
420 425 430
Leu Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His
435 440 445
His His
450
<210> 24
<211> 450
<212> PRT
<213> Artificial Sequence
<400> 24
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Pro Phe Arg Ser Asp Glu Ile Glu His Leu
260 265 270
Val Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu
275 280 285
Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His
290 295 300
Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn
305 310 315 320
Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp
325 330 335
Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys
340 345 350
His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro
355 360 365
Asp Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln
370 375 380
His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu
385 390 395 400
Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe
405 410 415
Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp
420 425 430
Leu Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His
435 440 445
His His
450
<210> 25
<211> 450
<212> PRT
<213> Artificial Sequence
<400> 25
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Pro Arg Ser Asp Glu Ile Glu His Leu
260 265 270
Val Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu
275 280 285
Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His
290 295 300
Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn
305 310 315 320
Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp
325 330 335
Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys
340 345 350
His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro
355 360 365
Asp Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln
370 375 380
His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu
385 390 395 400
Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe
405 410 415
Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp
420 425 430
Leu Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His
435 440 445
His His
450
<210> 26
<211> 450
<212> PRT
<213> Artificial Sequence
<400> 26
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Pro Ser Asp Glu Ile Glu His Leu
260 265 270
Val Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu
275 280 285
Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His
290 295 300
Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn
305 310 315 320
Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp
325 330 335
Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys
340 345 350
His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro
355 360 365
Asp Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln
370 375 380
His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu
385 390 395 400
Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe
405 410 415
Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp
420 425 430
Leu Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His
435 440 445
His His
450
<210> 27
<211> 450
<212> PRT
<213> Artificial Sequence
<400> 27
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Ser Pro Asp Glu Ile Glu His Leu
260 265 270
Val Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu
275 280 285
Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His
290 295 300
Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn
305 310 315 320
Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp
325 330 335
Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys
340 345 350
His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro
355 360 365
Asp Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln
370 375 380
His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu
385 390 395 400
Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe
405 410 415
Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp
420 425 430
Leu Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His
435 440 445
His His
450
<210> 28
<211> 450
<212> PRT
<213> Artificial Sequence
<400> 28
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Ser Asp Pro Glu Ile Glu His Leu
260 265 270
Val Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu
275 280 285
Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His
290 295 300
Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn
305 310 315 320
Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp
325 330 335
Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys
340 345 350
His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro
355 360 365
Asp Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln
370 375 380
His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu
385 390 395 400
Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe
405 410 415
Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp
420 425 430
Leu Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His
435 440 445
His His
450
<210> 29
<211> 450
<212> PRT
<213> Artificial Sequence
<400> 29
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Pro Ile Glu His Leu
260 265 270
Val Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu
275 280 285
Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His
290 295 300
Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn
305 310 315 320
Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp
325 330 335
Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys
340 345 350
His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro
355 360 365
Asp Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln
370 375 380
His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu
385 390 395 400
Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe
405 410 415
Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp
420 425 430
Leu Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His
435 440 445
His His
450
<210> 30
<211> 450
<212> PRT
<213> Artificial Sequence
<400> 30
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Pro Glu His Leu
260 265 270
Val Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu
275 280 285
Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His
290 295 300
Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn
305 310 315 320
Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp
325 330 335
Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys
340 345 350
His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro
355 360 365
Asp Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln
370 375 380
His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu
385 390 395 400
Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe
405 410 415
Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp
420 425 430
Leu Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His
435 440 445
His His
450
<210> 31
<211> 450
<212> PRT
<213> Artificial Sequence
<400> 31
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu Pro His Leu
260 265 270
Val Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu
275 280 285
Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His
290 295 300
Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn
305 310 315 320
Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp
325 330 335
Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys
340 345 350
His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro
355 360 365
Asp Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln
370 375 380
His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu
385 390 395 400
Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe
405 410 415
Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp
420 425 430
Leu Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His
435 440 445
His His
450
<210> 32
<211> 450
<212> PRT
<213> Artificial Sequence
<400> 32
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu His Pro Leu
260 265 270
Val Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu
275 280 285
Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His
290 295 300
Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn
305 310 315 320
Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp
325 330 335
Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys
340 345 350
His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro
355 360 365
Asp Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln
370 375 380
His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu
385 390 395 400
Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe
405 410 415
Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp
420 425 430
Leu Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His
435 440 445
His His
450
<210> 33
<211> 450
<212> PRT
<213> Artificial Sequence
<400> 33
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu His Leu Pro
260 265 270
Val Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu
275 280 285
Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His
290 295 300
Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn
305 310 315 320
Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp
325 330 335
Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys
340 345 350
His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro
355 360 365
Asp Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln
370 375 380
His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu
385 390 395 400
Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe
405 410 415
Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp
420 425 430
Leu Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His
435 440 445
His His
450
<210> 34
<211> 450
<212> PRT
<213> Artificial Sequence
<400> 34
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu His Leu Val
260 265 270
Pro Val Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu
275 280 285
Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His
290 295 300
Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn
305 310 315 320
Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp
325 330 335
Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys
340 345 350
His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro
355 360 365
Asp Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln
370 375 380
His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu
385 390 395 400
Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe
405 410 415
Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp
420 425 430
Leu Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His
435 440 445
His His
450
<210> 35
<211> 450
<212> PRT
<213> Artificial Sequence
<400> 35
Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro
1 5 10 15
Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp
20 25 30
Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val
35 40 45
Gly Tyr Ile Leu Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val
50 55 60
Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr
65 70 75 80
Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala
85 90 95
Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu
100 105 110
His Asn Pro Tyr Pro Asp Tyr Arg Trp Leu Arg Thr Val Lys Thr Thr
115 120 125
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro
130 135 140
Tyr Asp Arg Ser Leu His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser
145 150 155 160
Gly Val Ala Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr
165 170 175
Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Met Ser Cys Asp Ile Phe
180 185 190
Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu Thr Cys Gly
195 200 205
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys
210 215 220
Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp
225 230 235 240
Val Ser Met Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Lys
245 250 255
Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu His Leu Val
260 265 270
Val Pro Glu Glu Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu
275 280 285
Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His
290 295 300
Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn
305 310 315 320
Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp
325 330 335
Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys
340 345 350
His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro
355 360 365
Asp Gly Asn Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln
370 375 380
His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu
385 390 395 400
Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe
405 410 415
Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp
420 425 430
Leu Gly Leu Pro Asn Trp Gly Lys Tyr His His His His His His His
435 440 445
His His
450

Claims (7)

1. The preG modified protein of the rabies virus is characterized in that the preG modified protein is obtained by mutating proline of Ile268, his270 and Val273 of a qC connecting region in the rabies virus preG protein;
The amino acid sequence of preG protein after site mutation is shown in sequence tables SEQ ID NO.13, SEQ ID NO.15 and SEQ ID NO. 18.
2. Use of a preG modified protein of rabies virus of claim 1 in the preparation of a vaccine, inactivated vaccine, attenuated vaccine or viral vector vaccine.
3. The preparation method of the preG modified protein vaccine of the rabies virus is characterized by comprising the following steps:
1) Constructing a vaccine: synthesizing the preG modified protein sequence of claim 1 onto an expression vector, constructing a recombinant expression plasmid, and then transforming and transfecting to obtain preG modified protein of the soluble rabies virus;
2) Purifying: pretreating culture supernatant after preG modified protein vaccine centrifugation, performing EDTA-resistant and alkali-resistant Ni filler affinity chromatography, and performing ultrafiltration concentration dialysis treatment;
3) Vaccine finished product: and preparing the ultrafiltered vaccine stock solution and an adjuvant into a vaccine finished product.
4. The method for preparing preG modified protein vaccine according to claim 3, wherein the expression vector in step 1 includes a prokaryotic expression vector and a eukaryotic expression vector.
5. The method of claim 3, wherein the expression vector comprises an insect viral vector, a yeast expression vector, a CHO expression vector, or other viral expression vector.
6. The method for preparing a preG modified protein vaccine according to claim 3, wherein the expression plasmid comprises RV-preG-pFastbac-268, RV-preG-pFastbac-270, RV-preG-pFastbac-273.
7. The method for preparing preG modified protein vaccine according to claim 3, wherein concentrated purified rabies RVG-268p, RVG-270p and RVG-273p are respectively added with 201 adjuvant according to the ratio of 1:1.
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Structural Analysis of Rabies Virus Glycoprotein Reveals pH-Dependent Conformational Changes and Interactions with a Neutralizing Antibody;Fanli Yang等;《Cell Host Microbe》;第27卷(第3期);第445页右栏第一段、第442页左栏最后一段、第446页左栏第1-2段、第446页右栏第1段、图4、图5 *
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