CN114957408A - 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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CN114957408A
CN114957408A CN202110209081.6A CN202110209081A CN114957408A CN 114957408 A CN114957408 A CN 114957408A CN 202110209081 A CN202110209081 A CN 202110209081A CN 114957408 A CN114957408 A CN 114957408A
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CN114957408B (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 protein, and particularly relates to a preG modified protein of rabies virus and application thereof. The preG modified protein is obtained by mutating, inserting or substituting the amino acid of the qc junction region (Leu 257-Val 273) in the rabies virus preG protein into other amino acids, wherein the other amino acids comprise proline. The amino acid sequence of the preG modified protein comprises the amino acid sequences shown in SEQ ID NO. 2-SEQ ID NO.35 of the sequence table. The helix area of the qC area of the preG protein is stabilized, the phenomenon that the helix area forms an unnecessary helix structure to cause the folding of the qC area of the preG protein is prevented, and the unfolding and folding of the qC area to form a postG structure are prevented, so that the conformation of the preG is stabilized, the stability of the structure of the preG is ensured, the expression quantity of the protein structure is improved, meanwhile, the immunogenicity of the preG is also improved, and a vaccine which is large in expression quantity and can be expressed in a soluble mode 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 protein, and particularly relates to a preG modified protein of rabies virus and application thereof.
Background
Rabies is a 100% lethal human-animal comorbidity that seriously jeopardizes public health and life safety, and is caused by Rabies virus (Rabies virus, RABV) of lysssavir, a genus of Rabies virus in Rhabdovorodae, the family rhabdoviridae. The pathogeny causing rabies in China is mainly RABV, the number of RABV population types in China is 7 (China I-VI), the 7 th newly discovered population is from the case of the autonomous region in Tibet in 2017, the types and the number of epidemic populations are different from each other, for example, the Yunnan province in the border and the Hunan province in the traditional high-incidence region, and the number of the populations is as many as 4. At present, no effective treatment means exists, and vaccination of rabies vaccines is the only effective prevention measure.
The rabies vaccine in China is subjected to the stages of sheep brain, rabbit brain nerve tissue vaccine, cell culture concentrated vaccine, cell culture purified adjuvant-free vaccine and the like. The rabies vaccine for people in China only comprises hamster kidney cell vaccine and Vero cell purified vaccine. The virus of the domestic animal rabies live vaccine which is produced and marketed in China is introduced from abroad in 1979 and is a Flury strain rabies chick embryo low-generation virus (LEP) which is suitable for growing in a BHK21 cell line. Rabies, canine distemper, parainfluenza, adenopathy and parvovirus which are successfully developed in China in 2008 are put into a five-combination live vaccine and put on the market. Because the live vaccine virus is possibly subjected to recovery mutation in vivo to cause morbidity, potential safety hazards exist, so in recent years, the inactivated vaccine is gradually paid attention to, at present, the canine vaccine in the market of China mainly comprises the live vaccine and the inactivated vaccine, but the veterinary rabies inactivated vaccine is gradually replacing the attenuated live vaccine.
The RABV genome encodes 5 structural proteins, the nucleoprotein (N protein), the phosphoprotein (P protein), the matrix protein (M protein), the transmembrane glycoprotein (G protein) and the viral polymerase protein (L protein).
Apoptosis following RABV infection is primarily caused by G protein, and the less virulent the virus induces the more severe the apoptosis. The G protein is a trimeric transmembrane protein present on the surface of a virus envelope, is the only glycoprotein on the surface of RV virus, and is bound with a receptor on the surface of a cell. The G protein is the only surface antigen for stimulating the organism to generate a neutralizing antibody, and can also induce T cells and stimulate the organism to generate cellular immunity, thereby not only removing viruses in the Central Nervous System (CNS), but also inducing the nerve cells to generate apoptosis. Plays a key role in rabies virus pathogenesis and immunity. Therefore, the rabies virus G protein has wide application in the aspects of vaccine development, antibody detection and the like.
As viruses of the rhabdoviridae, the G proteins of VSV, RABV exist in two conformations: since the RABV G protein with a natural structure is in a preG state under alkaline conditions and in a postG state under acidic conditions, the structure of the RABV G protein is not stable. There are two structural states of the rabies G protein structure, the pre-fusion G protein (preG) of the viral surface bent hairpin structure and the post-fusion G protein (postG) mediating the membrane fusion extended conformation. The G protein on the surface of the virus exists mostly in a bent hairpin structure (preG) state.
To establish infection, RABV first binds to the host receptor and then enters the cell via the endocytic pathway. The endocytosome acidic environment triggers the inversion 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. RABV-G was thought to revert to its native preG conformation following transport through the acidic environment of the Golgi apparatus. This pH-dependent conformational change was also confirmed in the G protein of VSV viruses of the same Rhabdoviridae.
As the RV G protein with a natural structure is in a preG conformation under alkaline conditions and in a postG conformation under acidic conditions, the structure of the RV G protein is not stable. Meanwhile, the preG conformation can gradually convert to the postG conformation along with the change of the storage time and the stability, so that the antigenic sites of the preG conformation are reduced, and the immunogenicity is reduced. The structure of the antigen is used as a main antigen, and the immunogenicity of the antigen can be influenced, so that the immunogenicity of the antigen can be effectively improved by stabilizing the preG structure of the antigen, and the immune protection effect can be enhanced. Therefore, stabilizing the pre-fusion state of the viral membrane protein is a hot spot in the current vaccine research.
Disclosure of Invention
Aiming at the problems, the invention provides a preG modified protein of rabies virus and application thereof. The amino acid of qC connecting region of preG protein of rabies virus is mutated into proline, and the helical structure of Leu257-Val273 region of preG protein of rabies virus is destroyed, so that the protein structure expression quantity is improved, and the immunogenicity is also 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 (qC region and a CD region spiral region (Leu 257-Val 273)) in the preG protein of the rabies virus with other amino acids; the helix structure of the amino acid of the preG protein is damaged, so that the conformation of the preG protein is stabilized, and the preG protein cannot be changed into an expanded conformation from a folded hairpin conformation;
the other amino acids include proline.
The number of the amino terminal mutation or insertion sites comprises one or more.
The site of the amino acid mutation or insertion comprises one of the amino acids of the qC attachment region of the preG protein and the spiral region of the CD region (Leu 257-Val 273) to which qC is attached.
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 the amino acid of the preG protein after the site mutation comprises the amino acid sequence shown in SEQ ID NO. 2-SEQ ID NO.18 of the sequence table;
the amino acid sequence of the preG protein after the amino acid is inserted into the site comprises the amino acid sequence shown in SEQ ID NO. 19-SEQ ID NO.35 of the sequence table;
the invention also provides the application of the preG modified protein of the rabies virus in the aspects of vaccine development and antibody detection; is applied to preparing vaccines, inactivated vaccines, attenuated vaccines or virus vector vaccines.
The invention also provides a preparation method of the preG modified protein vaccine of the rabies virus, which comprises the following steps:
1) constructing a vaccine: synthesizing a preG modified protein sequence on an expression vector, constructing a recombinant expression plasmid, and then transforming and transfecting to obtain a preG modified protein vaccine of the soluble rabies virus;
2) and (5) 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 the steps of pretreating culture supernatant after centrifugation of the preG modified protein vaccine, performing EDTA and alkali resistant Ni filler affinity chromatography, and performing ultrafiltration concentration dialysis treatment;
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, adjusting the conductance to 10 ms/cm-50 ms/cm by using NaCl, adjusting the pH to 6.0-8.0, filtering by using a membrane below 0.45um, and using filtrate for chromatographic purification;
the operation of affinity chromatography comprises the following steps:
1) the histidine tag is designed at the end C of the upstream construction, so that high-purity target protein can be conveniently and quickly obtained, and the affinity chromatography filler is an EDTA and alkali resistant Ni filler; the concentration, the conductivity and the pH value of the filler balance liquid imidazole are the same as those of the pretreatment liquid: 10mM to 0.1M PB (Na) 2 HPO 4 ，NaH 2 PO 4 ) (ii) a The NaCl concentration is 50 mM-0.6M; the concentration of imidazole is 5-50 mM; pH6.2-8.0; the column height is 5-25 cm, and the flow rate is 30-300 cm/h; the equilibrium volume is 2 CV-20 CV;
2) after filtration, loading a sample, wherein the loading flow rate is 30-300 cm/h, and the loading amount is 2-10 mg per mL of filler;
3) after the sample is loaded, washing the column with a balance solution until the base line is flat, wherein the flow speed is 30-300 cm/h;
4) washing impurities, namely target protein with a histidine tag is bound on the column under the conditions, and part of the impurity protein is non-specifically adsorbed on the column, so that the non-specifically bound impurity protein can be washed by selecting proper conditions, the target protein cannot be washed under the conditions, the purity is ensured to be improved, and the recovery rate is not reduced;
the impurity washing buffer solution mainly comprises 10 mM-0.1M PB (Na) 2 HPO 4 ，NaH 2 PO 4 ) The NaCl concentration is 50 mM-0.6M, the imidazole concentration is 10-80 mM, the pH value is 6.2-8.0, and the flow rate is 60 cm/h-300 cm/h;
eluting, wherein after impurities are washed, the target protein is eluted by using an elution buffer solution, and the elution buffer solution comprises 10 mM-0.1M PB (Na) 2 HPO 4 ，NaH 2 PO 4 ) The NaCl concentration is 20 mM-0.6M, the imidazole concentration is 100 mM-1M, 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, the buffer solution needs to be replaced, and the ultrafiltration method is selected for carrying out the buffer solutionAnd the size of the ultrafiltration membrane package is 3.5-30 KD, and the ultrafiltration buffer solution comprises but is not limited to: 10mM to 0.1M PB (Na) 2 HPO 4 ，NaH 2 PO 4 ) 1-3% of mannitol, 1-4% of arginine hydrochloride and 6.0-8.0 of pH;
the process has the main characteristics of simple process route, high purity after purification and high recovery rate, wherein the purity after purification can reach more than 90 percent, and the recovery rate is more than 85 percent;
the sample after ultrafiltration is vaccine stock solution, and the vaccine stock solution and the adjuvant are prepared into a vaccine finished product together to form a final product.
The invention has the following beneficial effects:
the rabies virus preG modified protein stabilizes the spiral area of the qC connecting area (Leu 257-Val 273) of the preG protein by mutating, inserting or replacing other amino acids of the qC connecting area in the rabies virus preG protein, prevents the spiral area from forming redundant spiral structure to cause the folding of the qC connecting area, and prevents the qC connecting area from being unfolded to form a postG structure, thereby stabilizing the conformation of the preG, ensuring the stability of the structure, improving the expression quantity of the protein structure, simultaneously improving the immunogenicity of the protein structure, and forming a vaccine which has large expression quantity and is soluble and expressive;
the preG modified protein of the 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 the replication of the viruses is inhibited. The preG modified protein of rabies virus has wide application in the aspects of development of rabies virus vaccines, antibody detection and the like.
Drawings
FIG. 1 shows the results of PCR identification of 6 groups of recombinant plasmids of example 1;
FIG. 2 shows the results of PCR electrophoresis of 6 recombinant bacmid groups of example 1;
FIG. 3 shows the WB assay results of the expression of the P0 generation protein in example 1;
FIG. 4 shows WB assay results of the expression of the culture supernatant proteins in the reactor in example 1;
FIG. 5 shows SDS-PAGE of protein expression in example 1;
FIG. 6 shows WB detection results of the sample after protein purification of example 1;
FIG. 7 shows the results of experiments on the binding ability of rabies G protein mutants to preG and postG antibodies in different pH solutions of example 1;
FIG. 8 shows the results of IgG antibody level detection after immunization with different rabies preG modified protein vaccines of example 1;
FIG. 9 shows the results of the antibody neutralization experiments performed after immunization with the different rabies preG modified protein vaccines of example 1.
Detailed Description
The present invention is described in further detail in the following description of specific embodiments and the accompanying drawings, it is to be understood that these embodiments are merely illustrative of the present invention and are not intended to limit the scope of the invention, which is defined by the appended claims, and modifications thereof by those skilled in the art after reading this disclosure that are equivalent to the above described embodiments.
All the raw materials and reagents of the invention are conventional market raw materials and reagents unless otherwise specified.
Examples
Preparation of preG modified protein of rabies virus and vaccine thereof:
1. construction of recombinant plasmid
The preG protein consists of the following domains: the pre-G modified protein recombinant plasmid is characterized in that a membrane outer region (1-439 aa), a transmembrane region (440-461 aa) and a membrane inner region (462-550 aa) are inserted between any amino acid sites of a qC junction region (Leu 257-Val 273) of the membrane outer region, the pre-G protein is changed into an expanded conformation from a folded hairpin conformation, fusion is carried out in a tandem mode, a codon optimization system is adopted to design and synthesize the pre-G modified protein recombinant plasmid on a pFastBac1 carrier, any amino acid of 5 qC junction regions (Leu 257-Val 273) is mutated into proline, and the proline is respectively mutated into 260, 265, 268, 270 and 273 amino acids, plasmids RV-preG-pFastac-260, RV-preG-pFastac-265, RV-preG-pFastac-268, RV-preG-pFastac-270 and RV-preG-pFastac-273 are numbered respectively. Meanwhile, a plasmid RV-preG-pFastbac of the gene with the preG modified protein is constructed.
2. Construction of recombinant bacmids
Transforming recombinant plasmids RV-preG-pFastac, RV-preG-pFastac-260, RV-preG-pFastac-265, RV-preG-pFastac-268, RV-preG-pFastac-270 and RV-preG-pFastac-273 into escherichia coli DH10Bac competent cells, carrying out blue and white spot screening recombinants on an LB plate containing X-preGal/IPTPRG (shown in figure 2), selecting 5 white colonies from the recombinants, carrying out PCR identification, wherein the size of a target fragment is 800bp, the PCR identification result is shown in figure 1, and the 5 white single colonies all carry target genes, which indicates that the plasmids are expressed correctly;
2 PCR identified positive recombinants were picked for amplification culture, bacmid DNA was isolated using alkaline lysis method, and the results of bacmid electrophoresis are shown in FIG. 2, with higher bacmid concentration and correct band expression.
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 mu g of sterile bacmid into one of the two sterile centrifuge tubes, mixing uniformly, adding 0.5mL Grace's culture medium and 90 mu L EZ-trans transfection reagent into the other sterile centrifuge tube, and mixing uniformly; transferring all liquid in the centrifugal tube containing the transfection reagent into the centrifugal tube containing the bacmid, uniformly mixing, and standing for 15-20 minutes at room temperature to prepare a bacmid-carrier compound; taking out the cells from the biochemical shaker, adding the prepared bacmid-carrier compound while shaking, and placing the mixture back into the biochemical shaker for culture at 28 ℃ and 120rpm after shaking uniformly.
Virus harvesting and expression verification: taking appropriate amount of SF9 cells at exponential phase, diluting with fresh SF9 medium to make its density 1 × 10 6 Per mL, inoculated in 125mL shake flasks, 20 mL. And inoculating P0 virus infection according to MOI = 1-5, taking 0.5mL on the 4 th-5 th day to perform Western Blot experiment, wherein the result is shown in figure 3, and the results show that RV-preG-pFastac, RV-preG-pFastac-260, RV-preG-pFastac-265, RV-preG-pFastac-268, RV-preG-pFastac-270 and RV-preG-pFastac-273 all have protein expression on SF9 cells, and the expression forms are mainly culture supernatants.
Meanwhile, the virus is harvested on the 4 th to 5 th days and is stored at the temperature of minus 80 ℃.
Expanding culture in a reactor: taking appropriate amount of SF9 cells in exponential phase, diluting with fresh SF9 medium to make its density be 1 × 10 6 PermL, inoculated in 250mL shake flasks, 50mL, inoculated with P2 seed virus at MOI = 1-5, 28 ℃, 120 rpm. After 3-4 days of culture, according to seed toxicity: SF9 cell (density 1X 10) 6 /mL) =5% -10% inoculation expansion to 500 mL; after 3-4 days of culture, according to seed toxicity: SF9 cell (density 1X 10) 6 The culture volume is 5L, and the culture conditions are pH 6.5 +/-0.1, DO 40%, 28 ℃ and 100 rpm. And harvesting culture supernatant on 5-7 days of culture. The protein expression condition of the culture supernatant is detected by Western Blot experiment, and the result is shown in figure 4, which shows that the protein expression quantity of the reactor amplification culture is high, and the method can be used for the next purification experiment.
The expression of baculovirus-insect system expresses RVG-260p, RVG-265p, RVG-268p, RVG-270p and RVG-273p proteins respectively according to the operation method.
4. Purification of rabies preG modified protein subunit vaccine
(I) sample pretreatment
Taking 500mL of culture supernatant expressed by an insect rod system with 6 proteins, adding imidazole to a final concentration of 50mM, and fully stirring to completely dissolve after adding;
the conductivity of the above treated solution is adjusted to 40ms/cm by NaCl, the pH is adjusted to 7.0, the treated solution is filtered by a 0.45um membrane, and the filtrate is used for chromatographic purification.
(II) affinity chromatography
(1) The affinity chromatography filler is an EDTA and alkali resistant Ni filler; equilibrium solution, 20mM PB, NaCl concentration 0.3M, imidazole concentration 10mM, pH7.2; the height of the column is 5cm, and the flow rate is 30 cm/h; equilibrium volume 10 CV;
(2) after filtration, loading a sample, wherein the loading flow rate is 30cm/h, and the loading amount is 2-10 mg per mL of filler;
(3) after the sample is loaded, washing the column by using a balance solution until the base line is flat, wherein the flow rate is 30 cm/h;
(4) impurity washing: washing mixed buffer solution 20mM PB, NaCl concentration 0.3M, imidazole concentration 30mM, pH7.2, flow rate 30 cm/h;
(5) and (3) elution: elution buffer 20mM PB, NaCl concentration 0.3M, imidazole concentration 300mM, pH7.2, flow rate of 30 cm/h.
(III) Ultrafiltration dialysis
The affinity chromatography collection liquid has high concentration imidazoleAnd replacing the buffer solution by using an ultrafiltration method, wherein the size of an ultrafiltration membrane package is 10KD, and the ultrafiltration buffer solution: 20mMPB (Na) 2 HPO 4 ，NaH 2 PO 4 ) Mannitol 1.5%, arginine hydrochloride 2%, pH7.4, dialysis multiple 6 times volume. After dialysis, the mixture is concentrated to the concentration of 2 mg/mL;
the purity of RVG-260p, RVG-265p, RVG-268p, RVG-270p and RVG-273p after purification by the above method through Coomassie brilliant blue staining analysis is 93.1%, 92.8%, 93.2%, 93%, 92.5% and 93.5% respectively. The calculated recovery rates by content detection are 90.5%, 90.2%, 91%, 90.6%, 89.9% and 90.8%, respectively.
Identification of the purified protein: taking 15 mu L of each purified protein, mixing the protein with an equal amount of 2x electrophoresis sample-adding buffer solution, performing SDS-PAGE electrophoresis, wherein the electrophoresis result is shown in figure 5, one part of the protein is stained 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 purified test result almost has no other impurity band; and the other part is subjected to membrane conversion and Western blot identification, the WB detection result is shown in figure 6, and the molecular weight corresponding to the band of the sample in the lane 3 is the target molecular weight.
5. Antibody binding Capacity test of preG protein of different conformations
The murine anti-preG and postG antibodies were diluted to a concentration of 0.2. mu.g/ml with coating buffer (pH9.6) and coated overnight at 4 ℃. Then blocked with 5% skim milk in PBS for 1 hour at room temperature. After blocking, 50. mu.l of protein with a histidine tag at a concentration of 200. mu.g/ml were diluted 5-fold in gradient with RVG, RVG-260p, RVG-265p, RVG-268p, RVG-270p, RVG-273p in phosphate buffer pH5.5 and 8.0, respectively, added to the wells and incubated at 37 ℃ for one hour, then diluted anti-His mouse antibody was added at a ratio of 1:3000 for incubation for 1.5h, and goat anti-mouse IgG-HRP was added for incubation for 1 h. The reaction was carried out for 3 minutes while keeping out of the light by adding a developing solution, and after terminating with 2M HCl, the absorbance was read at 450 nm.
The results show that several proteins of RVG, RVG-260p, RVG-265p, RVG-268p, RVG-270p and RVG-273p can be combined with the antibody of the postG protein under different pH values, and the binding capacity is not different; however, as for the binding ability of the preG antibody, several proteins are not much different from the binding ability of the preG antibody under the condition of pH8.0; however, at pH5.5, the RVG-268p, RVG-270p and RVG-273p binding capacity is significantly higher than that of the original RVG and RVG-260p, indicating that the mutation of RVG-268p, RVG-270p and RVG-273p stabilizes the preG conformation, and the results are shown in FIG. 7.
The above proteins were used to bind to antibodies specific for preG and postG, respectively, and the results indicated that the mutated preG-modified protein was able to bind to both antibodies. The mutated RVG-268p, RVG-270p and RVG-273p modified proteins have no obvious reduction of the binding ability with the preG antibody under the condition of low pH value.
6. Preparation of subunit vaccines
The 'W/O/W' water-in-oil-in-water is adopted, the adjuvant is 201 adjuvant purchased from SEPPIC company of France, has an immune enhancement effect, can better improve cellular immunity, the 'water-in-oil-in-water' is the best adjuvant technology in inactivated vaccine, the anaphylactic reaction of water is small when the water is injected at the outermost layer, and the protection time is long because the second layer is oil; the third layer is water again, and the anaphylactic reaction is reduced when the third layer enters the body again. 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 respectively, and mixing uniformly with protein content of 100 μ g/ml.
7. Immunization with subunit vaccines
Mice were randomly divided into PBS control group and six protein-immunized groups of 16 mice each. The mice in each group are immunized by leg muscle 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, blood is collected from the tail vein at the 7 th and 14 d after the first needle immunization, and serum is separated and stored at the temperature of 80 ℃ below zero.
8. Immune mouse ELISA detection IgG antibody
Serum from mice 14 d after the initial immunization was taken and the IgG levels were detected using ELISA, according to the protocol of the IgG ELISA kit. The test data are shown in figure 8, and the vaccine groups RVG-265p, RVG-268p, RVG-270p and RVG-273p all produced higher antibody levels, while the vaccine groups RVG and RVG-260p produced 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 a fluorescent antibody by using a infected cell, and then measuring the half infection quantity (TCID 50) of the CVS-11 cell by using a fluorescent antibody staining method; sera were taken from 7d and 14 d mice after immunization and the level of neutralizing antibodies was determined. The detection data are shown in figure 9, and the neutralizing capacity of the RVG-265p, RVG-268p, RVG-270p and RVG-273p vaccine group antibodies is obviously stronger than that of the RVG, RVG-260p and vaccine group antibodies.
10. Toxicity attacking protection experiment
Mice were randomized into PBS control and RVG-265p, RVG-268p, RVG-270p, RVG-273p immunization groups of 16 mice each. 0.1 mL/mouse (protein content 10 ug/mouse) was intraperitoneally injected at 0 th and 7d, respectively. Mice were injected intracranially 14 d after the first immunization with a pre-titrated dose of 50 LD50 of CVS-11 virus, 0.03 mL each. The mice that died after the 5 th day and presented with typical rabies encephalopathy were counted and the survival rate was calculated by observing 14 d continuously. The mice in each experimental group were observed for abnormal conditions such as crinkles, pili, convulsions, and spasms, and for feeding, excretions, and mental manifestations of the mice. And (3) immunization result: the PBS control group began to develop clinical symptoms at the 7 th day of toxicity attack, which were manifested as disorders, curls, extreme excitement, etc., and died gradually at the 8 th to 12 th days. The weight of the mice in the vaccine group is not abnormal, and the mice do not have symptoms of rabies virus infection such as curling, mania and hair disorder, and all survive.
The preG modified protein can be applied to the aspects of vaccine development and antibody detection, the method for preparing the vaccine is not limited to the above, the preG modified protein can also be used for preparing inactivated vaccine, attenuated vaccine or virus vector vaccine, the expression vector for preparing the vaccine is not limited to the above, the preG modified protein also comprises a yeast expression vector, a CHO expression vector or a virus expression vector, the preparation method and the effect are similar to the above, and repeated details are not needed.
In conclusion, the preG modified protein vaccine of rabies virus can effectively neutralize free virus, inhibit the replication of the virus, form a vaccine with large expression amount and soluble expression, and improve the protein structure expression amount and the effective immunogenicity of the vaccine.
Sequence listing
<110> Guangzhou Yuanbo Biotechnology Co., Ltd
<120> preG modified protein of rabies virus and application thereof
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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
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 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
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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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Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp
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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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Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly Val Asp Leu
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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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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
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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
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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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Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn
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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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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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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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Leu Val Asn Pro 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
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
435 440 445
His
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<212> PRT
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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 (10)

1. The preG modified protein of the rabies virus is obtained by mutating or inserting other amino acids into the amino acids of a qC connecting region Leu257-Val273 in the rabies virus preG protein.
2. The preG-modified protein of claim 1, in which the other amino acids include proline.
3. The preG-modified protein of claim 1, where the number of sites for the amino-terminal mutation or insertion includes one or more.
4. The preG-modified protein of claim 1, where the site of amino acid mutation or insertion comprises one of the amino acids Leu257-Val273 of the qc junction region of the preG protein.
5. The preG-modified protein of claim 4, whose amino acid sequence includes SEQ ID No.2 to SEQ ID No.35 of the sequence listing.
6. Application of preG modified protein of rabies virus in vaccine development and antibody detection.
7. The preG modified protein of rabies virus is used in preparing vaccine, inactivated vaccine, attenuated vaccine or virus vector vaccine.
8. A preparation method of a preG modified protein vaccine of rabies virus is characterized by comprising the following steps:
1) constructing a vaccine: synthesizing a preG modified protein sequence on an expression vector, constructing a recombinant expression plasmid, and then transforming and transfecting to obtain a preG modified protein vaccine of the soluble rabies virus;
2) and (5) purifying.
9. The method for preparing a preG-modified protein vaccine as defined in claim 8, wherein the expression vector of step 1 includes a prokaryotic expression vector and a eukaryotic expression vector.
10. The method for preparing a preG-modified protein vaccine as defined in claim 9, wherein the expression vector includes an insect virus vector, a yeast expression vector, a CHO expression vector, or other viral expression vectors.
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Citations (2)

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Publication number Priority date Publication date Assignee Title
CN1635905A (en) * 2001-07-20 2005-07-06 乔治亚大学研究基金会 Attenuated rabies virus with nucleoprotein mutation at the phosphorylation site for vaccination against rabies and gene therapy in the CNS
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Publication number Priority date Publication date Assignee Title
CN1635905A (en) * 2001-07-20 2005-07-06 乔治亚大学研究基金会 Attenuated rabies virus with nucleoprotein mutation at the phosphorylation site for vaccination against rabies and gene therapy in the CNS
CN103087155A (en) * 2013-01-20 2013-05-08 复旦大学 Rabies virus vaccine enhancin, gene for coding enhancin and application of enhancin

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ANNA FERLIN等: "Characterization of pH-sensitive molecular switches that trigger the structural transition of vesicular stomatitis virus glycoprotein from the postfusion state toward the prefusion state", JOURNAL OF VIROLOGY, vol. 88, no. 22, pages 13397 *
B VOLLMER等: "The prefusion structure of herpes simplex virus glycoprotein B", SCIENCE ADVANCES, vol. 6, no. 39, pages 208 - 211 *
FANLI YANG等: "Structural Analysis of Rabies Virus Glycoprotein Reveals pH-Dependent Conformational Changes and Interactions with a Neutralizing Antibody", 《CELL HOST MICROBE》, vol. 27, no. 3, pages 1 - 2 *
左利民等: "α-螺旋跨膜蛋白的折叠和自组装", 科学通报, no. 15, pages 2 *

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