CN101622272A - Modified flagellin improved toll-like receptor 5 stimulating activity - Google Patents

Modified flagellin improved toll-like receptor 5 stimulating activity Download PDF

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CN101622272A
CN101622272A CN200880004041A CN200880004041A CN101622272A CN 101622272 A CN101622272 A CN 101622272A CN 200880004041 A CN200880004041 A CN 200880004041A CN 200880004041 A CN200880004041 A CN 200880004041A CN 101622272 A CN101622272 A CN 101622272A
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李浚行
李施恩
金守永
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CHONNAM NAT UNIVERSITY
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Abstract

Disclosed herein are flagellin mutants having an enhanced activity of stimulating the toll-like receptor-5 (hereinafter referred to as ''TLR5''). More specifically, disclosed are flagellin mutants, prepared by point-mutating some of the amino acids of a TRL5 agonist flagellin so as to enhance the TRL-stimulating activity of the flagellin.

Description

The flagellin of the modification of improvement toll sample acceptor 5 stimulating activities
Technical field
The present invention relates to have the flagellin mutant of enhanced toll sample receptor-5 (hereinafter referred to as " TLR5 ") stimulating activity, particularly the flagellin mutant for preparing with the TLR5 stimulating activity that strengthens flagellin by some amino acid of point mutation TRL5 agonist flagellin.
Background technology
Flagellum is the important structure element of decision bacterial motility, and it is made up of crozier, matrix and filament usually.Known flagellum helps to move about or cluster motion, bacterium taxis, pathogenic micro-organism to the adhesion and the biomembranous formation of host cell.The protein subunit that forms the flagellum filament is called as flagellin, and flagellin assembles the formation filament regularly.Hayashi etc. disclose the TLR5 that expresses in the mammalian cell and can discern the flagellin of Gram-negative and gram positive bacterium and activate NF-κ B (Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng JK, Akira S, Underhill DM, AderemA:Nature 410:1099-1103,2001).
Toll sample acceptor (TLRs) is typical " pattern recognition acceptor (PRRs) ", its identification is present in " pathogenic agent associated molecular pattern (PAMPs) " in the pathogenic agent, and not only in Mammals, found this receptor, also found this receptor on the surface of insect and vegetable cell.So far found 13 kinds of TLRs, this has guided energetically to the research of TLRs agonist (Takeuchi O:Cell 124 (4): 783-801,2006 for Akira S, Uematsu S).
PRRs (for example TLRs) is distributed in the surface or tenuigenin of host cell, and after being subjected to multiple PAMPs stimulation, PRRs can induce innate immunity to reply, and regulates adaptive immune response.Thereby the TLR agonist can be used as the target spot that is suitable for developing panimmunity conditioning agent (especially vaccine adjuvant).
In this article, term " vaccine adjuvant " refers to can strengthen, prolong or quicken the material by vaccine antigen inductive Ag-specific immune response with the vaccine co-administered time.The vaccine adjuvant that approval is used for human body comprises aluminum phosphate, aluminium hydroxide and squalene emulsifying agent.Vaccine adjuvant must satisfy at least one in following five requirements: 1) regulate costimulatory molecules in the lip-deep expression of antigen presenting cell, inducing antigen-specific T lymphocyte responses, or immunoregulation (for example secretion of the regulating cell factor); 2) antigen presentation; 3) induce the CD8+ cytotoxic T lymphocyte to reply; 4) targeting; With 5) form and store.
Ideal vaccine adjuvant: must be safe 1); 2) must be biodegradable in vivo; Compare when 3) individually dosed, must show that effectively preventative or therapeutic immunization is replied with antigen; 4) must be material through chemistry or biometric authentication; 5) preferably when being lower than antigen, concentration works; With 6) must have the long transformation period, so that they can easily be used for commerce or clinical application.
Maybe can consider to comprise as vaccine adjuvant at present: 1) mineral salt, for example aluminum hydroxide gel as the material of vaccine adjuvant; 2) surfactant materials; 3) material of bacterial derivation; 4) cytokine or hormone; 5) polyanion; 6) polypropylene-base; 7) carrier; 8) contain viral live body carrier; With 9) vehicle, for example mineral grease plastid.Wherein, at present just comprised vibrio cholerae (Vibrio cholerae) deutero-Toxins,exo-, cholera (CT) and intestinal bacteria (Escherichia coli) deutero-heat-labile toxins (LT) by active research and the protein derived vaccine adjuvant that receives very big concern.These vaccine adjuvants are induced in mucous membrane district and blood plasma and are produced antigen-specific antibodies according to reports, and induce B7-2 in the expression on antigen presenting cell (APCs) surface to stimulate the costimulatory signal of CD4+ helper cell.But these adjuvants are to have high enterotoxication extracellular toxin, thereby are carrying out about toxicity that reduces them and the research that strengthens their adjuvanticity.
As disclosed among the open text WO 2005/070455 of pct international patent, the inventor has made up adhesion and the invasion factor of transposon mutant body library with screening Vibrio vulnificus (Vibrio vulnificus), and having analyzed the Tn flanking region of losing three clones of the adhesion of host cell and mobility, the inventor has differentiated two flagellum operons that contain 56 genes as a result.In this analytic process, find that Vibrio vulnificus always has six flagellin genes (flaA, flaB, flaF, flaC, flaD and flaE), wherein the flaB gene is the main ingredient of this flagellin.The inventor has studied the polar flagellum protein ingredient of Vibrio vulnificus---and flagellin recombinant protein (FlaB) can be used as the possibility at the composition vaccine of Vibrio vulnificus, the inventor has found flagellin recombinant protein (FlaB) except that having high antigenicity as a result, also has strong vaccine adjuvant and renders a service.For explaining this discovery, the inventor has carried out further research.The result has proved when the vaccine antigen Toxoid,tetanus is applied to the experimental animal nasal cavity with flagellin, described flagellin has enlarged vaccine potency by the TLR5 that signal is passed to host cell with activating immune system, when the tetanus toxin of lethal dose is attacked by using tetanus toxin and flagellin mice immunized, described flagellin has been induced the immunity of defence completely of contratoxin, show that described flagellin has excellent mucosal vaccine adjuvants and renders a service (Lee SE, Kim SY, JeongBC, Kim YR, Bae SJ, Ahn OS, Lee JJ, Song HC, Kim JM, Choy HE, Chung SS, Kweon MN, Rhee JH.:Infect.Immun.74:694-702,2006).
In multiple TLR agonist, the flagellin that stimulates TLR5 is that a kind of protein that is different from other TLR agonists (CpG-DNA, MLP (mycoplasma lipopeptid)) is formed.Thereby, can composite character can in addition, can be made up multiple recombination fusion protein by the reorganization flagellin of Sustainable Control with enhanced TLR5 stimulating activity.
According to result of study to the flagellin three-dimensional structure, at flagellin monomer Semi-polarity amino-acid residue and charge residue residue and those polare Aminosaeren residues in other monomer and the reaction of charge residue residue, cause the axial interaction between the generation monomer and form polymer, thereby form characteristic filar structure (the Yonekura K of flagellin, Maki-Yonekura S, Namba K:Complete atomic model of the bacterial flagellar filament by electroncryomicroscopy.Nature.2003 424 (6949): 643-50; Samatey FA, Imada K, Nagashima S, Vonderviszt F, Kumasaka T, Yamamoto M, Namba K:Structure ofthe bacterial flagellar protofilament and implications for a switch for supercoiling.Nature.2001 410 (6826): 331-7).Research according to Smith etc., the flagellin polymer of TLR5 nonrecognition filament type, but identification flagellin monomer (Smith KD, Andersen-Nissen E, Hayashi F, Strobe K, Bergman MA, Barrett SL, Cookson BT, Aderem A.Toll-likereceptor 5 recognizes a conserved site on flagellin required for protofilamentformation and bacterial motility.Nat Immunol.2003 4 (12): 1247-53).
Observed common issue with is that these vaccines lack the vaccine adjuvant that effectively can enlarge associated responses specifically in the preventative and therapeutic vaccine (vaccine of transmissible disease, autoimmune disorder, anaphylactic disease and cancer) that uses in clinical application at present.Thereby, need to develop safer and more virtuous vaccine adjuvant consumingly.
Summary of the invention
Correspondingly, the inventor participates in the reorganization flagellin mutant that axial interactional amino-acid residue has prepared Vibrio vulnificus flagellin gene flaB by changing expection, this mutant can be suppressed at the polarity charge reaction that relates in the axial interaction between the flagellin monomer, and the flagellin mutant that has been found that preparation is compared with existing (wild-type) flagellin has remarkable enhanced TLR5 stimulating activity, thereby has finished the present invention.
Therefore, the purpose of this invention is to provide flagellin mutant with enhanced TLR5 stimulating activity.
Another object of the present invention provides and contains the vaccine adjuvant of at least a described flagellin mutant as activeconstituents.
For achieving the above object, the invention provides and be used for being suppressed at interactional flagellin mutant between the TLR5 agonist flagellin flagellin monomer.
The present invention also provides and contains the vaccine adjuvant of at least a described flagellin mutant as activeconstituents.
In the present invention, compare the flagellin vaccine adjuvant that the flagellin mutant with enhanced TLR5 stimulating activity has been developed improvement with existing flagellin by providing, the flagellin vaccine adjuvant of having found this improvement demonstrates effective mucosal vaccine adjuvants effectiveness by stimulating TLR5, as disclosed among the open text No.WO 2005/070455 of pct international patent.Flagellin mutant according to the present invention will be applied to treating transmissible disease, autoimmune disorder, and anaphylactic disease or the like, in this external anticancer therapy, described flagellin mutant will provide the important step that connects fundamental research and clinical study.Thereby flagellin mutant of the present invention is applied in the multiple vaccine preparation can create very high added value.
Description of drawings
Fig. 1 has shown the comparison of the amino acid identity between Salmonellas (Salmonella) flagellin St-FliC, coli flagellum albumen Ec-FliC and the Vibrio vulnificus flagellin Vv-FlaB, sequence in its center is flagellin structural domain (the Samatey FA of each bacterial strain, Deng the people., Structure of thebacterial flagellar protofilament and implications for a switch for supercoiling.Nature.2001 410 (6826): 331-7).
Fig. 2 is presented to make up in the flagellin mutant process of the present invention, on Vibrio vulnificus flagellin gene flaB, induce synoptic diagram (the Samatey FA of the strategy of rite-directed mutagenesis, Deng the people, Structure ofthe bacterial flagellar protofilament and implications for a switch for supercoiling.Nature.2001 410 (6826): 331-7).
Fig. 3 and 4 shows that reorganization flagellin mutant of the present invention influences the flagellin polymeric in the aqueous solution.
Fig. 5 and 6 is the charts of measuring result that show the TLR stimulating activity of flagellin mutant of the present invention.
Embodiment
Hereinafter the present invention will be described in more detail.
The present invention relates to the flagellin mutant, this flagellin mutant is that some amino acid by point mutation TLR5 agonist flagellin prepares, so that the flagellin sudden change suppresses the monomeric poly cooperation of flagellin usefulness, thereby this flagellin mutant shows enhanced TLR5 stimulating activity.
The present invention relates to: the flagellin mutant (D70A) shown in the SEQ ID NO:2, it is to become L-Ala (A) to prepare by aspartic acid (D70) site-directed mutagenesis that wild-type Vibrio vulnificus flagellin FlaB is last the 70th, and described according to one's analysis flagellin FlaB has participated in the axial interaction between the flagellin monomer in the TLR5 agonist flagellin; Flagellin mutant (R93A) shown in the SEQ ID NO:4, it is by becoming L-Ala (A) to prepare arginine (R93) site-directed mutagenesis on the 93rd; Flagellin mutant (L97W) shown in the SEQ ID NO:6, it is by becoming tryptophane (W) to prepare leucine (L97) site-directed mutagenesis on the 97th; Flagellin mutant (N101A) shown in the SEQ ID NO:8, it is by becoming L-Ala (A) to prepare l-asparagine (N101) site-directed mutagenesis on the 101st; Flagellin mutant (S103W) shown in the SEQ ID NO:10, it is by becoming tryptophane (W) to prepare Serine (S103) site-directed mutagenesis on the 103rd; Flagellin mutant (E108A) shown in the SEQ ID NO:12, it is by becoming L-Ala (A) to prepare L-glutamic acid (E108) site-directed mutagenesis on the 108th; Flagellin mutant (N135D) shown in the SEQ ID NO:14, it is by becoming aspartic acid (D) to prepare l-asparagine (N135) site-directed mutagenesis on the 135th; Flagellin mutant (A151W) shown in the SEQ ID NO:16, it is by becoming tryptophane (W) to prepare L-Ala (A151) site-directed mutagenesis on the 151st; Flagellin mutant (N153W) shown in the SEQ ID NO:18, it is by becoming tryptophane (W) to prepare l-asparagine (N153) site-directed mutagenesis on the 153rd; With the flagellin mutant (V291W) shown in the SEQ IDNO:20, it is by becoming tryptophane (W) to prepare Xie Ansuan (V291) site-directed mutagenesis on the 291st.In addition, among the flagellin mutant, flagellin mutant with two or more aminoacid replacement also within the scope of the invention, the example can comprise the flagellin mutant (D70A/N135D) shown in the SEQ ID NO:44, it is by aspartic acid (D70) site-directed mutagenesis on the 70th of the wild-type Vibrio vulnificus flagellin FlaB is become L-Ala (A) and becomes aspartic acid (D) to prepare l-asparagine (N135) site-directed mutagenesis on the 135th, but scope of the present invention is not limited thereto.
Term " flagellin " refers to constitute the individual molecule of the flagellum that determines bacterial motility herein.
Flagellin mutant provided by the present invention has remarkable enhanced TLR5 stimulating activity, and this phenomenon is the poly cooperation usefulness of comparing and having reduced flagellin significantly with existing flagellin mutant owing to described flagellin mutant, thereby suppresses the flagellin polymerization.
Correspondingly, flagellin mutant with remarkable enhanced TLR5 stimulating activity prepared in accordance with the present invention can provide more effective TLR agonist replacing existing flagellin, has the vaccine adjuvant that enhanced is renderd a service thereby provide to compare with existing flagellin.
Prove that the method that flagellin mutant of the present invention has an enhanced TLR5 stimulating activity may further comprise the steps:
1) the flagellin mutant shown in the preparation SEQ ID NO:2,4,6,8,10,12,14,16,18,20 and 44;
2) confirm that the flagellin mutant is suppressed at the polymerization of flagellin in the aqueous solution; With
3) confirm respectively the recombinate TLR5 activation of flagellin mutant of in intestinal bacteria inductive.
Embodiment
Hereinafter the present invention is described in more detail with reference to embodiment and test implementation example.But it should be understood that these embodiment and test implementation example only are to illustrate the present invention, rather than limit the scope of the invention.
The feature of bacterial strain uses therefor of the present invention and plasmid is summed up in following table 1.The preparation method of every kind of flagellin mutant and the concrete property of each flagellin mutant in embodiment and test implementation example, have been shown.
[table 1]
Bacterial strain or plasmid Feature The source
Intestinal bacteria
??DH5α F recA1; Restricted feminine gender This laboratory
??ER2566 ??F-λ-fhuA2[lon]ompT?lacZ::T7gene1?gal ??sulA11 ??(mcrC-mrr)114::IS10R(mcr-73::miniTn10-TetS)2 ??R(zgb-210::Tn10)(TetS)endA1[dcm] ??New?England ??Biolab
??BL21(DE3) ??F-ompT?hsdS B(r B-m B-)gal?dcm(DE3) ??Novagen
Plasmid
??pCR2.1 ??topO PCR TA cloning vector, Amp R，Km R ??Invitrogen
??pTYB12 IMPACT (intein mediation with affine chitin combination tag purifying) expression vector, Amp R ??New?England ??Biolab
The cultivation of each bacterial strain and preservation
Following examples and the used intestinal bacteria of test implementation example are cultivated in Luria Bertani substratum (DifcoCo.).This cultivation bacterial strain is kept at-80 ℃ of refrigerators in 30% glycerine.
Embodiment 1: by site-directed mutagenesis preparation reorganization flagellin mutant
Analytical data (Yonekura K according to the Salmonellas flagellin three-dimensional structure of former report, Maki-Yonekura S, Namba K:Complete atomic model of the bacterial flagellarfilament by electron cryomicroscopy.Nature.2003424 (6949): 643-50; SamateyFA, Imada K, Nagashima S, Vonderviszt F, Kumasaka T, Yamamoto M, Namba K:Structure of the bacterial flagellar protofilament and implications for a switch forsupercoiling.Nature.2001410 (6826): 331-7), with the bioinformatic analysis that the inventor carries out, the needed amino-acid residue that axially interacts between the Vibrio vulnificus flagellin FlaB monomer is as follows.Promptly be respectively and participated in axial interactional each monomeric L-Ala (A151) of going up the 153rd of the aspartic acid (D70) of the 70th of subunit (upper subunit), the l-asparagine (D135) of going up the 135th of subunit and last subunit, by respectively with the l-asparagine (N100) of the 100th of each monomeric subunit (lower subunit) down, down the 108th of subunit L-glutamic acid (E108), the polarity charge of the L-Ala (A292) of the 292nd of the arginine (R93) of the 93rd of subunit and following subunit reacts and forms polymer down.The polarity charge reaction promptly takes place between following amino-acid residue: go up flagellin D70 and following flagellin N100; Last flagellin N135 and following flagellin E107; Last flagellin A151 and following flagellin R93; With last flagellin N153 and following flagellin A292.
In the present invention, by carrying out site-directed mutagenesis and prepare following flagellin mutant to participating between the flagellin subunit axially interactional following amino-acid residue according to one's analysis: the flagellin mutant (D70A) shown in the SEQ ID NO:2, it is by becoming L-Ala (A) to prepare aspartic acid (D70) site-directed mutagenesis on the 70th; Flagellin mutant (R93A) shown in the SEQ ID NO:4, it is by becoming L-Ala (A) to prepare arginine (R93) site-directed mutagenesis on the 93rd; Flagellin mutant (N101A) shown in the SEQ ID NO:8, it is by becoming L-Ala (A) to prepare l-asparagine (N101) site-directed mutagenesis on the 101st; Flagellin mutant (E108A) shown in the SEQ ID NO:12, it is by becoming L-Ala (A) to prepare L-glutamic acid (E108) site-directed mutagenesis on the 108th; Flagellin mutant (N135D) shown in the SEQ ID NO:14, it is by becoming aspartic acid (D) to prepare l-asparagine (N135) site-directed mutagenesis on the 135th; Flagellin mutant (A151W) shown in the SEQ ID NO:16, it is by becoming tryptophane (W) to prepare L-Ala (A151) site-directed mutagenesis on the 151st; With the flagellin mutant (N153W) shown in the SEQ ID NO:18, it is by becoming tryptophane (W) to prepare l-asparagine (N153) site-directed mutagenesis on the 153rd.Yet the L-Ala (A292) that has participated in according to one's analysis on axial interactional the 292nd between the flagellin monomer is not used in the structure mutant, even also can not change because axially interact after this amino-acid residue is suddenlyd change.
In addition, by carrying out site-directed mutagenesis and prepare following mutant to infer participating in axial interactional following amino-acid residue between the flagellin monomer: the flagellin mutant (L97W) shown in the SEQ ID NO:6, it is by becoming tryptophane (W) to prepare leucine (L97) site-directed mutagenesis on the 97th; Flagellin mutant (S103W) shown in the SEQ ID NO:10, it is by becoming tryptophane (W) to prepare Serine (S103) site-directed mutagenesis on the 103rd; With the flagellin mutant (V291W) shown in the SEQ ID NO:20, it is by becoming tryptophane (W) to prepare Xie Ansuan (V291) site-directed mutagenesis on the 291st.
At first, in order to obtain to be used for the dna profiling of site-directed mutagenesis, adopt the PCR primers F laB-V5 shown in the SEQ ID NO:21 (5 '- GcggccgcAtggcagtgaatgtaaatgtaaatacaaac-3 '; The line part: the NotI restriction enzyme enzyme recognition site that is used to clone) and the PCR primers F laB-V4 shown in the SEQ ID NO:22 (5 '- CccgggGcctagtagacttagcgctga-3 '; Line part: the SmaI restriction enzyme enzyme recognition site that is used to clone), contain 1 of flaB gene ORF, 142-bp dna fragmentation by pcr amplification.Carry out pcr amplification under the following conditions with primers F laB-V5 and FlaB-V4: 95 ℃ of initial sex change 1 minute, 95 ℃ of sex change in 30 seconds, 70 ℃ of annealing were extended and were carried out 30 circulations in 1 minute in 30 seconds and 72 ℃ then, then 72 ℃ of last extensions 10 minutes.The flaB dna fragmentation of amplification is cloned in the PCR 2.1-TOPO cloning vector (Invitrogen Co.), and the carrier that makes is called " pCMM270 ".The pCMM270 plasmid is used for follow-up site-directed mutagenesis as dna profiling.
Be used to make up the PCR condition of Vibrio vulnificus flagellin gene flaB directed mutants and clone's plasmid is summed up in the following Table 2.
[table 2]
The mutational site Primer (SEQ ID NO) The PCR annealing temperature PCR2.1-top O clone's title PTYB12 clone's title
??D70A ??FlaB-SD1(SEQ?ID?NO：23) ??5′-gtacgtaacgccaacgcaggtatctcaatc-3′ ??FlaB-SD2(SEQ?ID?NO：24) ??5′-gattgagatacctgcgttggcgttacgtac-3′ ??63℃ ??pCMM272 ??pCMM276
??R93A ??FlaB-SD13-2(SEQ?ID?NO：25) ??5′-catcctacaacgtatggctgacctatctctacaatc-3′ ??FlaB-SD14-2(SEQ?ID?NO：26) ??5′-gattgtagagataggtcagccatacgttgtaggatg-3′ ??63℃ ??pCMM281 ??pCMM291
??L97W ??FlaB-SD17(SEQ?ID?NO：27) ??5′-gcgtgacctatcttggcaatccgcgaacgg-3′ ??FlaB-SD18(SEQ?ID?NO：28) ??5′-ccgttcgcggattgccaagataggtcacgc-3′ ??66℃ ??pCMM282 ??pCMM292
??N101A ??FlaB-SD9-3(SEQ?ID?NO：29) ??5′-ctacaatccgcggccggctcaaactcaaaatc-3′ ??FlaB-SD10-3(SEQ?ID?NO：30) ??5′-gattttgagtttgagccggccgcggattgtag-3′ ??64℃ ??pCMM283 ??pCMM293
??S103W ??FlaB-SD15(SEQ?ID?NO：31) ??5′-caatccgcgaacggctggaactcaaaatcag-3′ ??FlaB-SD16(SEQ?ID?NO：32) ??5′-ctgattttgagttccagccgttcgcggattg-3′ ??63℃ ??pCMM284 ??pCMM294
??E108A ??FlaB-SD11(SEQ?ID?NO：33) ??5′-caaactcaaaatcagcgcgcgtggcgattc-3′ ??FlaB-SD12(SEQ?ID?NO：34) ??5′-gaatcgccacgcgcgctgattttgagtttg-3′ ??63℃ ??pCMM285 ??pCMM295
??N135D ??FlaB-SD3(SEQ?ID?NO：35) ??5′-cgtcttttggtggtgacaagctgctaaacg-3′ ??FlaB-SD4(SEQ?ID?NO：36) ??5′-cgtttagcagcttgtcaccaccaaaagacg-3′ ??63℃ ??pCMM273 ??pCMM277
??A151W ??FlaB-SD5(SEQ?ID?NO：37) ??5′-gcaatgcaaattggttgggataacggtgaagcg-3′ ??FlaB-SD6(SEQ?ID?NO：38) ??5′-cgcttcaccgttatcccaaccaatttgcattgc-3′ ??63℃ ??pCMM286 ??pCMM296
??N153W ??FlaB-SD7(SEQ?ID?NO：39) ??5′-caaattggtgcggattggggtgaagcggtcatg-3′ ??FlaB-SD8(SEQ?ID?NO：40) ??5′-catgaccgcttcaccccaatccgcaccaatttg-3′ ??67℃ ??pCMM287 ??pCMM297
??V291W ??FlaB-SD19(SEQ?ID?NO：41) ??5′-gcgcaagagtcgtgggcgattgtggatgcg-3′ ??FlaB-SD20(SEQ?ID?NO：42) ??5′-cgcatccacaatcgcccacgactcttgcgc-3′ ??67℃ ??pCMM288 ??pCMM298
??D70A/N ??135D ??FlaB-SD1(SEQ?ID?NO：23) ??5′-gtacgtaacgccaacgcaggtatctcaatc-3′ ??FlaB-SD2(SEQ?ID?NO：23) ??5′-gattgagatacctgcgttggcgttacgtac-3′ ??63℃ ??pCMM274 ??pCMM278
In order to make up each directed mutants flaB DNAs (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D), adopt as above the primer of each shown in the table 2 to, with pCMM270 as pcr template with use fidelity Pfu polysaccharase (Stratagene Co.) to carry out the PCR reaction and come synthetic sudden change chain by supplier's explanation.Carry out each PCR reaction under the following conditions: 95 ℃ of initial sex change 45 seconds, 95 ℃ of sex change 45 seconds then, annealing was extended and was carried out 16 circulations in 10 minutes in 1 minute and 68 ℃ under temperature shown in the table 2, then 68 ℃ of last extensions 10 minutes.With SEQ ID NO:1,3,5,7,9,11,13,15,17 and 19 each the amplification rite-directed mutagenesis flaB DNAs be cloned in the PCR 2.1-TOPO cloning vector, the carrier that obtains is called " pCMM272 ", " pCMM281 ", " pCMM282 ", " pCMM283 ", " pCMM284 ", " pCMM285 ", " pCMM273 ", " pCMM286 ", " pCMM287 " and " pCMM288 " (table 2).
The two rite-directed mutagenesis flagellin D70A/N135D of D70A/N135D for shown in the preparation SEQ ID NO:44 carry out the PCR reaction with the pCMM273 that contains flaB DNA as template.Carry out PCR with PCR primers F laB-SD2 shown in the PCR primers F laB-SD1 shown in the SEQID NO:23, the SEQ ID NO:24 and fidelity Pfu polysaccharase (Stratagene Co.) by supplier's explanation and synthesize sudden change chain (D70A).Carry out PCR reaction under the following conditions: 95 ℃ of initial sex change 45 seconds, 95 ℃ of sex change 45 seconds then, extended in 1 minute and 68 ℃ 63 ℃ of annealing and to carry out 16 circulations in 10 minutes, then 68 ℃ of last extensions 10 minutes.The two rite-directed mutagenesis flaB DNA of the D70A/N135D of the amplification shown in the SEQ ID NO:43 are called " pCMM274 ".
With restriction enzyme NotI and SmaI digestion SEQ ID NO:1, each dna fragmentation that contains rite-directed mutagenesis flaB gene (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D) of 3,5,7,9,11,13,15,17 and 19, be cloned into then in the pTYB12 plasmid (NewEngland Biolabs) with the digestion of same restrictions restriction endonuclease.The plasmid that obtains is called " pCMM276 ", " pCMM291 ", " pCMM292 ", " pCMM293 ", " pCMM293 ", " pCMM294 ", " pCMM295 ", " pCMM277 ", " pCMM296 ", " pCMM297 ", " pCMM298 " and " pCMM278 ".These plasmids are transformed among the intestinal bacteria ER2566 by electricity, and to the expression of the 5-bromo-indoles that wherein adds 0.5mM-3-chloro-isopropyl-(IPTG) with the induced mutation flagellin gene.Use 1, the 4-dithiothreitol (DTT) (1,4-DTT) and chitin magnetic bead post from the intein fusion rotein, obtain point mutation flagellin (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D) by supplier's explanation.Before the use, use AffinityPak TMDetoxi Gel TMIntracellular toxin is eliminated gel (Pirece) and remove the intracellular toxin that contains in isolating point mutation albumen.Use Superdex 120 posts (AKTA-Prime Amersham) carries out gel-filtration to isolating recombinant protein, thus the SEQ IDNO:2 of purification of high-purity, 4,6,8,10,12,14,16,18, the reorganization flagellin mutant shown in 20 and 44.
Test implementation example 1: generation and its biochemical characteristic in the aqueous solution of reorganization flagellin mutant
In order to prepare reorganization flagellin mutant, each gene of coding flagellin mutant shown in the SEQ ID NO:1,3,5,7,9,11,13,15,17,19 and 43 all is cloned in the pTYB12 plasmid, then with plasmid transfection in intestinal bacteria ER2566.In substratum, add 0.3mMIPTG (sec.-propyl-β-D-1-thiogalactoside) to induce the generation of reorganization flagellin mutant, analyze the albumen that obtains by SDS-polyacrylamide gel electrophoresis and natural gel electrophoresis then.Analytical results is shown in Fig. 3 and 4.
Fig. 3 and 4 has shown the SDS-PAGE analytical results of wild-type flagellin (FlaB) and sudden change flagellin FlaB, shows among the figure that SEQ ID NO:2,4,6,8,10,12,14,16,18,20 and 44 point mutation flagellin (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D) and wild-type flagellin (FlaB) all have main band at the 41.5kDa place.In natural gel electrophoresis result, can observe wild-type FlaB albumen and form huge polymer, make most of albumen be retained in and concentrate in the glue, and some FlaB albumen that enter in the separation gel are distributed between 66kDa and the 140kDa.Yet point mutation flagellin of the present invention (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D) has formed the polymer of about 140kDa.The above results shows that wild-type FlaB albumen has formed huge polymer in the aqueous solution, but the polymer that the polymer that point mutation flagellin mutant of the present invention (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D) forms forms less than the wild-type flagellin.Therefore, can determine that the flagellin mutant of the present invention (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D) shown in the SEQ IDNO:2,4,6,8,10,12,14,16,18,20 and 44 has suppressed the polymerization of flagellin.
Test implementation example 2: the TLR5 stimulating activity of flagellin mutant
In order to measure the transcriptional activity of the NF-κ B that participates in TLR5-mediation signal,, press 1x10 to determine the TLR5 stimulating activity of the flagellin mutant of the present invention shown in wild-type FlaB and the SEQ ID NO:2,4,6,8,10,12,14,16,18,20 and 44 5The density of cells/well is planted in each hole of 24 orifice plates 293T cell branch and overnight incubation.Then, the reporter plasmid NF-κ B-Luc that uses FuGENE6 (Roche) that transcriptional activity will be observed (is taught by Jeong-Mok Kim, Instituteof Biomedical Science, Hanyang University College of Medicine, Korea S provides), the p3xFlag-hTLR5 plasmid of clone TLR5 gene is (by Dr.Steven B.Mizel, Departments ofMicrobiology and Immunology, Wake Forest University School of Medicine, USA provides) and beta galactose glycosides expression plasmid (Clontech) simultaneously in the transfered cell.After cell was cultivated 24 hours again, change fresh medium, and handled cell 18-24 hour with the flagellin mutant (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D) shown in the embodiment 1 isolating SEQ ID NO:2,4,6,8,10,12,14,16,18,20 and 44.Then, detect transcribing of NF-κ B in the cell by measure uciferase activity with luminometer (Berthold), measuring result shows in Fig. 5 and 6.As illustrated in Figures 5 and 6, compare with the control group of only handling with phosphoric acid buffer with the group that 20ng/ml wild-type FlaB albumen is handled, TLR5 mediation NF-κ B transcriptional activity has strengthened about 6.6 times.And compare with the control group of only handling with phosphoric acid buffer with the group that the flagellin FlaB mutant (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D) of whenever recombinating of 20ng/ml is handled, the NF-κ B transcriptional activity of TLR5 mediation has strengthened about 24.6 times, 14 times, 25 times, 22.6 times, 23.7 times, 21 times, 21.7 times, 22.5 times, 24.5 times, 16.6 times and 14.9 times respectively.
Sequence list text none
SEQ ID NO:1 and 2 is gene and aminoacid sequences of flagellin mutant (D70A), and this flagellin mutant is by becoming L-Ala (A) to prepare aspartic acid (D70) site-directed mutagenesis on the 70th of the Vibrio vulnificus flagellin FlaB.
SEQ ID NO:3 and 4 is gene and aminoacid sequences of flagellin mutant (R93A), and this flagellin mutant is by becoming L-Ala (A) to prepare arginine (R93) site-directed mutagenesis on the 93rd of the Vibrio vulnificus flagellin FlaB.
SEQ ID NO:5 and 6 is gene and aminoacid sequences of flagellin mutant (L97W), and this flagellin mutant is by becoming tryptophane (W) to prepare leucine (L97) site-directed mutagenesis on the 97th of the Vibrio vulnificus flagellin FlaB.
SEQ ID NO:7 and 8 is gene and aminoacid sequences of flagellin mutant (N101A), and this flagellin mutant is by becoming L-Ala (A) to prepare l-asparagine (N101) site-directed mutagenesis on the 101st of the Vibrio vulnificus flagellin FlaB.
SEQ ID NO:9 and 10 is gene and aminoacid sequences of flagellin mutant (S103W), and this flagellin mutant is by becoming tryptophane (W) to prepare Serine (S103) site-directed mutagenesis on the 103rd of the Vibrio vulnificus flagellin FlaB.
SEQ ID NO:11 and 12 is gene and aminoacid sequences of flagellin mutant (E108A), and this flagellin mutant is by becoming L-Ala (A) to prepare L-glutamic acid (E108) site-directed mutagenesis on the 108th of the Vibrio vulnificus flagellin FlaB.
SEQ ID NO:13 and 14 is gene and aminoacid sequences of flagellin mutant (N135D), and this flagellin mutant is by becoming aspartic acid (D) to prepare l-asparagine (N135) site-directed mutagenesis on the 135th of the Vibrio vulnificus flagellin FlaB.
SEQ ID NO:15 and 16 is gene and aminoacid sequences of flagellin mutant (A151W), and this flagellin mutant is by becoming tryptophane (W) to prepare L-Ala (A151) site-directed mutagenesis on the 151st of the Vibrio vulnificus flagellin FlaB.
SEQ ID NO:17 and 18 is gene and aminoacid sequences of flagellin mutant (N153W), and this flagellin mutant is by becoming tryptophane (W) to prepare l-asparagine (N153) site-directed mutagenesis on the 153rd of the Vibrio vulnificus flagellin FlaB.
SEQ ID NO:19 and 20 is gene and aminoacid sequences of flagellin mutant (V291W), and this flagellin mutant is by becoming tryptophane (W) to prepare Xie Ansuan (V291) rite-directed mutagenesis on the 291st of the Vibrio vulnificus flagellin FlaB.
SEQ ID NO:21-42 is the primer sequence that is used for the flagellin mutant shown in the SEQ ID NO:2,4,6,8,10,12,14,16,18,20 and 44.
SEQ ID NO:43 and 44 is gene and aminoacid sequences of flagellin mutant (D70A/N135D), and this flagellin mutant is by becoming aspartic acid (D70) rite-directed mutagenesis on the 70th of the Vibrio vulnificus flagellin FlaB L-Ala (A) and becoming aspartic acid (D) to prepare l-asparagine (N135) site-directed mutagenesis on the 135th of the Vibrio vulnificus flagellin FlaB.
Sequence table
＜110〉Chonnam Nat University (INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY)
＜120〉flagellin of the modification of improvement toll sample acceptor 5 stimulating activities
<150>KR10-2007-0013846
<151>2007-02-09
<150>KR10-2008-0011330
<151>2008-02-04
<160>44
<170>KopatentIn?1.71
<210>1
<211>1134
<212>DNA
＜213〉Vibrio vulnificus D70A FlaB gene
<400>1
atggcagtga?atgtaaatac?aaacgtagca?gcaatgacag?cacagcgtta?cctgaataac????60
gcaaacagcg?cacaacaaac?ttcgatggag?cgtctgtctt?caggtttcaa?aatcaacagt????120
gcaaaagatg?acgcagccgg?tctgcaaatc?tctaaccgct?tgaacgtaca?aagtcgcggt????180
ctagacgttg?cggtacgtaa?cgccaacgca?ggtatctcaa?tcgcacaaac?cgcagaaggt????240
gcgatgaacg?agaccaccaa?catcctacaa?cgtatgcgtg?acctatctct?acaatccgcg????300
aacggctcaa?actcaaaatc?agagcgcgtg?gcgattcaag?aagaagtgac?agcattgaat????360
gacgagctaa?accgtattgc?agaaaccacg?tcttttggtg?gtaacaagct?gctaaacggt????420
acttacggca?cgaaagcaat?gcaaattggt?gcggataacg?gtgaagcggt?catgctttca????480
ctgaaagaca?tgcgctctga?caacgtgatg?atgggcggcg?tgagctacca?agctgaagaa????540
ggcaaagaca?agaactggaa?tgtggccgca?ggcgacaacg?acttgacgat?tgcactgaca????600
gacagctttg?gtaacgagca?agagatcgaa?atcaacgcga?aagcgggtga?tgacatcgaa????660
gagctagcga?cgtacatcaa?cggtcaaact?gaccttgtaa?aagcgtcagt?gggtgaaggc????720
ggcaagctac?agatctttgc?tggtaacaac?aaagttcaag?gtgaaattgc?tttctcaggt????780
agcctagctg?gtgaacttgg?cctaggcgaa?ggcaaaaacg?tcacggtaga?cacgattgac????840
gtgacaaccg?tacaaggtgc?gcaagagtcg?gtagcgattg?tggatgcggc?actgaaatac????900
gtagacagcc?accgtgcaga?gctgggtgca?ttccagaacc?gtttcaacca?tgcaatcagc????960
aacttggaca?acatcaacga?aaacgtgaac?gcgtcgaaga?gccgaatcaa?agataccgac????1020
ttcgcgaaag?aaacgactca?gttgaccaag?acacaaattc?tatcgcaagc?atcaagttcc????1080
attcttgcgc?aagcgaaaca?agcgccaaac?tcagcgctaa?gtctactagg?ctaa??????????1134
<210>2
<211>377
<212>PRT
＜213〉Vibrio vulnificus D70A FlaB amino acid
<400>2
Met?Ala?Val?Asn?Val?Asn?Thr?Asn?Val?Ala?Ala?Met?Thr?Ala?Gln?Arg
1???????????????5??????????????????10??????????????????15
Tyr?Leu?Asn?Asn?Ala?Asn?Ser?Ala?Gln?Gln?Thr?Ser?Met?Glu?Arg?Leu
20??????????????????25??????????????????30
Ser?Ser?Gly?Phe?Lys?Ile?Asn?Ser?Ala?Lys?Asp?Asp?Ala?Ala?Gly?Leu
35??????????????????40??????????????????45
Gln?Ile?Ser?Asn?Arg?Leu?Asn?Val?Gln?Ser?Arg?Gly?Leu?Asp?Val?Ala
50??????????????????55??????????????????60
Val?Arg?Asn?Ala?Asn?Ala?Gly?Ile?Ser?Ile?Ala?Gln?Thr?Ala?Glu?Gly
65??????????????????70??????????????????75??????????????????80
Ala?Met?Asn?Glu?Thr?Thr?Asn?Ile?Leu?Gln?Arg?Met?Arg?Asp?Leu?Ser
85??????????????????90??????????????????95
Leu?Gln?Ser?Ala?Asn?Gly?Ser?Asn?Ser?Lys?Ser?Glu?Arg?Val?Ala?Ile
100?????????????????105?????????????????110
Gln?Glu?Glu?Val?Thr?Ala?Leu?Asn?Asp?Glu?Leu?Asn?Arg?Ile?Ala?Glu
115?????????????????120?????????????????125
Thr?Thr?Ser?Phe?Gly?Gly?Asn?Lys?Leu?Leu?Asn?Gly?Thr?Tyr?Gly?Thr
130?????????????????135?????????????????140
Lys?Ala?Met?Gln?Ile?Gly?Ala?Asp?Asn?Gly?Glu?Ala?Val?Met?Leu?Ser
145?????????????????150?????????????????155?????????????????160
Leu?Lys?Asp?Met?Arg?Ser?Asp?Asn?Val?Met?Met?Gly?Gly?Val?Ser?Tyr
165?????????????????170?????????????????175
Gln?Ala?Glu?Glu?Gly?Lys?Asp?Lys?Asn?Trp?Asn?Val?Ala?Ala?Gly?Asp
180?????????????????185?????????????????190
Asn?Asp?Leu?Thr?Ile?Ala?Leu?Thr?Asp?Ser?Phe?Gly?Asn?Glu?Gln?Glu
195?????????????????200?????????????????205
Ile?Glu?Ile?Asn?Ala?Lys?Ala?Gly?Asp?Asp?Ile?Glu?Glu?Leu?Ala?Thr
210?????????????????215?????????????????220
Tyr?Ile?Asn?Gly?Gln?Thr?Asp?Leu?Val?Lys?Ala?Ser?Val?Gly?Glu?Gly
225?????????????????230?????????????????235?????????????????240
Gly?Lys?Leu?Gln?Ile?Phe?Ala?Gly?Asn?Asn?Lys?Val?Gln?Gly?Glu?Ile
245?????????????????250?????????????????255
Ala?Phe?Ser?Gly?Ser?Leu?Ala?Gly?Glu?Leu?Gly?Leu?Gly?Glu?Gly?Lys
260?????????????????265?????????????????270
Asn?Val?Thr?Val?Asp?Thr?Ile?Asp?Val?Thr?Thr?Val?Gln?Gly?Ala?Gln
275?????????????????280?????????????????285
Glu?Ser?Val?Ala?Ile?Val?Asp?Ala?Ala?Leu?Lys?Tyr?Val?Asp?Ser?His
290?????????????????295?????????????????300
Arg?Ala?Glu?Leu?Gly?Ala?Phe?Gln?Asn?Arg?Phe?Asn?His?Ala?Ile?Ser
305?????????????????310?????????????????315?????????????????320
Asn?Leu?Asp?Asn?Ile?Asn?Glu?Asn?Val?Asn?Ala?Ser?Lys?Ser?Arg?Ile
325?????????????????330?????????????????335
Lys?Asp?Thr?Asp?Phe?Ala?Lys?Glu?Thr?Thr?Gln?Leu?Thr?Lys?Thr?Gln
340?????????????????345?????????????????350
Ile?Leu?Ser?Gln?Ala?Ser?Ser?Ser?Ile?Leu?Ala?Gln?Ala?Lys?Gln?Ala
355?????????????????360?????????????????365
Pro?Asn?Ser?Ala?Leu?Ser?Leu?Leu?Gly
370?????????????????375
<210>3
<211>1134
<212>DNA
＜213〉Vibrio vulnificus R93A FlaB gene
<400>3
atggcagtga?atgtaaatac?aaacgtagca?gcaatgacag?cacagcgtta?cctgaataac????60
gcaaacagcg?cacaacaaac?ttcgatggag?cgtctgtctt?caggtttcaa?aatcaacagt????120
gcaaaagatg?acgcagccgg?tctgcaaatc?tctaaccgct?tgaacgtaca?aagtcgcggt????180
ctagacgttg?cggtacgtaa?cgccaacgac?ggtatctcaa?tcgcacaaac?cgcagaaggt????240
gcgatgaacg?agaccaccaa?catcctacaa?cgtatggctg?acctatctct?acaatccgcg????300
aacggctcaa?actcaaaatc?agagcgcgtg?gcgattcaag?aagaagtgac?agcattgaat????360
gacgagctaa?accgtattgc?agaaaccacg?tcttttggtg?gtaacaagct?gctaaacggt????420
acttacggca?cgaaagcaat?gcaaattggt?gcggataacg?gtgaagcggt?catgctttca????480
ctgaaagaca?tgcgctctga?caacgtgatg?atgggcggcg?tgagctacca?agctgaagaa????540
ggcaaagaca?agaactggaa?tgtggccgca?ggcgacaacg?acttgacgat?tgcactgaca????600
gacagctttg?gtaacgagca?agagatcgaa?atcaacgcga?aagcgggtga?tgacatcgaa????660
gagctagcga?cgtacatcaa?cggtcaaact?gaccttgtaa?aagcgtcagt?gggtgaaggc????720
ggcaagctac?agatctttgc?tggtaacaac?aaagttcaag?gtgaaattgc?tttctcaggt????780
agcctagctg?gtgaacttgg?cctaggcgaa?ggcaaaaacg?tcacggtaga?cacgattgac????840
gtgacaaccg?tacaaggtgc?gcaagagtcg?gtagcgattg?tggatgcggc?actgaaatac????900
gtagacagcc?accgtgcaga?gctgggtgca?ttccagaacc?gtttcaacca?tgcaatcagc????960
aacttggaca?acatcaacga?aaacgtgaac?gcgtcgaaga?gccgaatcaa?agataccgac????1020
ttcgcgaaag?aaacgactca?gttgaccaag?acacaaattc?tatcgcaagc?atcaagttcc????1080
attcttgcgc?aagcgaaaca?agcgccaaac?tcagcgctaa?gtctactagg?ctaa??????????1134
<210>4
<211>377
<212>PRT
＜213〉Vibrio vulnificus R93A FlaB amino acid
<400>4
Met?Ala?Val?Asn?Val?Asn?Thr?Asn?Val?Ala?Ala?Met?Thr?Ala?Gln?Arg
1???????????????5??????????????????10??????????????????15
Tyr?Leu?Asn?Asn?Ala?Asn?Ser?Ala?Gln?Gln?Thr?Ser?Met?Glu?Arg?Leu
20??????????????????25??????????????????30
Ser?Ser?Gly?Phe?Lys?Ile?Asn?Ser?Ala?Lys?Asp?Asp?Ala?Ala?Gly?Leu
35??????????????????40??????????????????45
Gln?Ile?Ser?Asn?Arg?Leu?Asn?Val?Gln?Ser?Arg?Gly?Leu?Asp?Val?Ala
50??????????????????55??????????????????60
Val?Arg?Asn?Ala?Asn?Asp?Gly?Ile?Ser?Ile?Ala?Gln?Thr?Ala?Glu?Gly
65??????????????????70??????????????????75??????????????????80
Ala?Met?Asn?Glu?Thr?Thr?Asn?Ile?Leu?Gln?Arg?Met?Ala?Asp?Leu?Ser
85??????????????????90??????????????????95
Leu?Gln?Ser?Ala?Asn?Gly?Ser?Asn?Ser?Lys?Ser?Glu?Arg?Val?Ala?Ile
100?????????????????105?????????????????110
Gln?Glu?Glu?Val?Thr?Ala?Leu?Asn?Asp?Glu?Leu?Asn?Arg?Ile?Ala?Glu
115?????????????????120?????????????????125
Thr?Thr?Ser?Phe?Gly?Gly?Asn?Lys?Leu?Leu?Asn?Gly?Thr?Tyr?Gly?Thr
130?????????????????135?????????????????140
Lys?Ala?Met?Gln?Ile?Gly?Ala?Asp?Asn?Gly?Glu?Ala?Val?Met?Leu?Ser
145?????????????????150?????????????????155?????????????????160
Leu?Lys?Asp?Met?Arg?Ser?Asp?Asn?Val?Met?Met?Gly?Gly?Val?Ser?Tyr
165?????????????????170?????????????????175
Gln?Ala?Glu?Glu?Gly?Lys?Asp?Lys?Asn?Trp?Asn?Val?Ala?Ala?Gly?Asp
180?????????????????185?????????????????190
Asn?Asp?Leu?Thr?Ile?Ala?Leu?Thr?Asp?Ser?Phe?Gly?Asn?Glu?Gln?Glu
195?????????????????200?????????????????205
Ile?Glu?Ile?Asn?Ala?Lys?Ala?Gly?Asp?Asp?Ile?Glu?Glu?Leu?Ala?Thr
210?????????????????215?????????????????220
Tyr?Ile?Asn?Gly?Gln?Thr?Asp?Leu?Val?Lys?Ala?Ser?Val?Gly?Glu?Gly
225?????????????????230?????????????????235?????????????????240
Gly?Lys?Leu?Gln?Ile?Phe?Ala?Gly?Asn?Asn?Lys?Val?Gln?Gly?Glu?Ile
245?????????????????250?????????????????255
Ala?Phe?Ser?Gly?Ser?Leu?Ala?Gly?Glu?Leu?Gly?Leu?Gly?Glu?Gly?Lys
260?????????????????265?????????????????270
Asn?Val?Thr?Val?Asp?Thr?Ile?Asp?Val?Thr?Thr?Val?Gln?Gly?Ala?Gln
275?????????????????280?????????????????285
Glu?Ser?Val?Ala?Ile?Val?Asp?Ala?Ala?Leu?Lys?Tyr?Val?Asp?Ser?His
290?????????????????295?????????????????300
Arg?Ala?Glu?Leu?Gly?Ala?Phe?Gln?Asn?Arg?Phe?Asn?His?Ala?Ile?Ser
305?????????????????310?????????????????315?????????????????320
Asn?Leu?Asp?Asn?Ile?Asn?Glu?Asn?Val?Asn?Ala?Ser?Lys?Ser?Arg?Ile
325?????????????????330?????????????????335
Lys?Asp?Thr?Asp?Phe?Ala?Lys?Glu?Thr?Thr?Gln?Leu?Thr?Lys?Thr?Gln
340?????????????????345?????????????????350
Ile?Leu?Ser?Gln?Ala?Ser?Ser?Ser?Ile?Leu?Ala?Gln?Ala?Lys?Gln?Ala
355?????????????????360?????????????????365
Pro?Asn?Ser?Ala?Leu?Ser?Leu?Leu?Gly
370?????????????????375
<210>5
<211>1134
<212>DNA
＜213〉Vibrio vulnificus L97W FlaB gene
<400>5
atggcagtga?atgtaaatac?aaacgtagca?gcaatgacag?cacagcgtta?cctgaataac????60
gcaaacagcg?cacaacaaac?ttcgatggag?cgtctgtctt?caggtttcaa?aatcaacagt????120
gcaaaagatg?acgcagccgg?tctgcaaatc?tctaaccgct?tgaacgtaca?aagtcgcggt????180
ctagacgttg?cggtacgtaa?cgccaacgac?ggtatctcaa?tcgcacaaac?cgcagaaggt????240
gcgatgaacg?agaccaccaa?catcctacaa?cgtatgcgtg?acctatcttg?gcaatccgcg????300
aacggctcaa?actcaaaatc?agagcgcgtg?gcgattcaag?aagaagtgac?agcattgaat????360
gacgagctaa?accgtattgc?agaaaccacg?tcttttggtg?gtaacaagct?gctaaacggt????420
acttacggca?cgaaagcaat?gcaaattggt?gcggataacg?gtgaagcggt?catgctttca????480
ctgaaagaca?tgcgctctga?caacgtgatg?atgggcggcg?tgagctacca?agctgaagaa????540
ggcaaagaca?agaactggaa?tgtggccgca?ggcgacaacg?acttgacgat?tgcactgaca????600
gacagctttg?gtaacgagca?agagatcgaa?atcaacgcga?aagcgggtga?tgacatcgaa????660
gagctagcga?cgtacatcaa?cggtcaaact?gaccttgtaa?aagcgtcagt?gggtgaaggc????720
ggcaagctac?agatctttgc?tggtaacaac?aaagttcaag?gtgaaattgc?tttctcaggt????780
agcctagctg?gtgaacttgg?cctaggcgaa?ggcaaaaacg?tcacggtaga?cacgattgac????840
gtgacaaccg?tacaaggtgc?gcaagagtcg?gtagcgattg?tggatgcggc?actgaaatac????900
gtagacagcc?accgtgcaga?gctgggtgca?ttccagaacc?gtttcaacca?tgcaatcagc????960
aacttggaca?acatcaacga?aaacgtgaac?gcgtcgaaga?gccgaatcaa?agataccgac????1020
ttcgcgaaag?aaacgactca?gttgaccaag?acacaaattc?tatcgcaagc?atcaagttcc????1080
attcttgcgc?aagcgaaaca?agcgccaaac?tcagcgctaa?gtctactagg?ctaa??????????1134
<210>6
<211>377
<212>PRT
＜213〉Vibrio vulnificus L97W FlaB amino acid
<400>6
Met?Ala?Val?Asn?Val?Asn?Thr?Asn?Val?Ala?Ala?Met?Thr?Ala?Gln?Arg
1???????????????5??????????????????10??????????????????15
Tyr?Leu?Asn?Asn?Ala?Asn?Ser?Ala?Gln?Gln?Thr?Ser?Met?Glu?Arg?Leu
20??????????????????25??????????????????30
Ser?Ser?Gly?Phe?Lys?Ile?Asn?Ser?Ala?Lys?Asp?Asp?Ala?Ala?Gly?Leu
35??????????????????40??????????????????45
Gln?Ile?Ser?Asn?Arg?Leu?Asn?Val?Gln?Ser?Arg?Gly?Leu?Asp?Val?Ala
50??????????????????55??????????????????60
Val?Arg?Asn?Ala?Asn?Asp?Gly?Ile?Ser?Ile?Ala?Gln?Thr?Ala?Glu?Gly
65??????????????????70??????????????????75??????????????????80
Ala?Met?Asn?Glu?Thr?Thr?Asn?Ile?Leu?Gln?Arg?Met?Arg?Asp?Leu?Ser
85??????????????????90??????????????????95
Trp?Gln?Ser?Ala?Asn?Gly?Ser?Asn?Ser?Lys?Ser?Glu?Arg?Val?Ala?Ile
100?????????????????105?????????????????110
Gln?Glu?Glu?Val?Thr?Ala?Leu?Asn?Asp?Glu?Leu?Asn?Arg?Ile?Ala?Glu
115?????????????????120?????????????????125
Thr?Thr?Ser?Phe?Gly?Gly?Asn?Lys?Leu?Leu?Asn?Gly?Thr?Tyr?Gly?Thr
130?????????????????135?????????????????140
Lys?Ala?Met?Gln?Ile?Gly?Ala?Asp?Asn?Gly?Glu?Ala?Val?Met?Leu?Ser
14?????????????????515?????????????????0155?????????????????160
Leu?Lys?Asp?Met?Arg?Ser?Asp?Asn?Val?Met?Met?Gly?Gly?Val?Ser?Tyr
165?????????????????170?????????????????175
Gln?Ala?Glu?Glu?Gly?Lys?Asp?Lys?Asn?Trp?Asn?Val?Ala?Ala?Gly?Asp
180?????????????????185?????????????????190
Asn?Asp?Leu?Thr?Ile?Ala?Leu?Thr?Asp?Ser?Phe?Gly?Asn?Glu?Gln?Glu
195?????????????????200?????????????????205
Ile?Glu?Ile?Asn?Ala?Lys?Ala?Gly?Asp?Asp?Ile?Glu?Glu?Leu?Ala?Thr
210?????????????????215?????????????????220
Tyr?Ile?Asn?Gly?Gln?Thr?Asp?Leu?Val?Lys?Ala?Ser?Val?Gly?Glu?Gly
225?????????????????230?????????????????235?????????????????240
Gly?Lys?Leu?Gln?Ile?Phe?Ala?Gly?Asn?Asn?Lys?Val?Gln?Gly?Glu?Ile
245?????????????????250?????????????????255
Ala?Phe?Ser?Gly?Ser?Leu?Ala?Gly?Glu?Leu?Gly?Leu?Gly?Glu?Gly?Lys
260?????????????????265?????????????????270
Asn?Val?Thr?Val?Asp?Thr?Ile?Asp?Val?Thr?Thr?Val?Gln?Gly?Ala?Gln
275?????????????????280?????????????????285
Glu?Ser?Val?Ala?Ile?Val?Asp?Ala?Ala?Leu?Lys?Tyr?Val?Asp?Ser?His
290?????????????????295?????????????????300
Arg?Ala?Glu?Leu?Gly?Ala?Phe?Gln?Asn?Arg?Phe?Asn?His?Ala?Ile?Ser
305?????????????????310?????????????????315?????????????????320
Asn?Leu?Asp?Asn?Ile?Asn?Glu?Asn?Val?Asn?Ala?Ser?Lys?Ser?Arg?Ile
325?????????????????330?????????????????335
Lys?Asp?Thr?Asp?Phe?Ala?Lys?Glu?Thr?Thr?Gln?Leu?Thr?Lys?Thr?Gln
340?????????????????345?????????????????350
Ile?Leu?Ser?Gln?Ala?Ser?Ser?Ser?Ile?Leu?Ala?Gln?Ala?Lys?Gln?Ala
355?????????????????360?????????????????365
Pro?Asn?Ser?Ala?Leu?Ser?Leu?Leu?Gly
370?????????????????375
<210>7
<211>1134
<212>DNA
＜213〉Vibrio vulnificus N101A FlaB gene
<400>7
atggcagtga?atgtaaatac?aaacgtagca?gcaatgacag?cacagcgtta?cctgaataac????60
gcaaacagcg?cacaacaaac?ttcgatggag?cgtctgtctt?caggtttcaa?aatcaacagt????120
gcaaaagatg?acgcagccgg?tctgcaaatc?tctaaccgct?tgaacgtaca?aagtcgcggt????180
ctagacgttg?cggtacgtaa?cgccaacgac?ggtatctcaa?tcgcacaaac?cgcagaaggt????240
gcgatgaacg?agaccaccaa?catcctacaa?cgtatgcgtg?acctatctct?acaatccgcg????300
gccggctcaa?actcaaaatc?agagcgcgtg?gcgattcaag?aagaagtgac?agcattgaat????360
gacgagctaa?accgtattgc?agaaaccacg?tcttttggtg?gtaacaagct?gctaaacggt????420
acttacggca?cgaaagcaat?gcaaattggt?gcggataacg?gtgaagcggt?catgctttca????480
ctgaaagaca?tgcgctctga?caacgtgatg?atgggcggcg?tgagctacca?agctgaagaa????540
ggcaaagaca?agaactggaa?tgtggccgca?ggcgacaacg?acttgacgat?tgcactgaca????600
gacagctttg?gtaacgagca?agagatcgaa?atcaacgcga?aagcgggtga?tgacatcgaa????660
gagctagcga?cgtacatcaa?cggtcaaact?gaccttgtaa?aagcgtcagt?gggtgaaggc????720
ggcaagctac?agatctttgc?tggtaacaac?aaagttcaag?gtgaaattgc?tttctcaggt????780
agcctagctg?gtgaacttgg?cctaggcgaa?ggcaaaaacg?tcacggtaga?cacgattgac????840
gtgacaaccg?tacaaggtgc?gcaagagtcg?gtagcgattg?tggatgcggc?actgaaatac????900
gtagacagcc?accgtgcaga?gctgggtgca?ttccagaacc?gtttcaacca?tgcaatcagc????960
aacttggaca?acatcaacga?aaacgtgaac?gcgtcgaaga?gccgaatcaa?agataccgac????1020
ttcgcgaaag?aaacgactca?gttgaccaag?acacaaattc?tatcgcaagc?atcaagttcc????1080
attcttgcgc?aagcgaaaca?agcgccaaac?tcagcgctaa?gtctactagg?ctaa??????????1134
<210>8
<211>377
<212>PRT
＜213〉Vibrio vulnificus N101A FlaB amino acid
<400>8
Met?Ala?Val?Asn?Val?Asn?Thr?Asn?Val?Ala?Ala?Met?Thr?Ala?Gln?Arg
1???????????????5??????????????????10??????????????????15
Tyr?Leu?Asn?Asn?Ala?Asn?Ser?Ala?Gln?Gln?Thr?Ser?Met?Glu?Arg?Leu
20??????????????????25??????????????????30
Ser?Ser?Gly?Phe?Lys?Ile?Asn?Ser?Ala?Lys?Asp?Asp?Ala?Ala?Gly?Leu
35??????????????????40??????????????????45
Gln?Ile?Ser?Asn?Arg?Leu?Asn?Val?Gln?Ser?Arg?Gly?Leu?Asp?Val?Ala
50??????????????????55??????????????????60
Val?Arg?Asn?Ala?Asn?Asp?Gly?Ile?Ser?Ile?Ala?Gln?Thr?Ala?Glu?Gly
65??????????????????70??????????????????75??????????????????80
Ala?Met?Asn?Glu?Thr?Thr?Asn?Ile?Leu?Gln?Arg?Met?Arg?Asp?Leu?Ser
85??????????????????90??????????????????95
Leu?Gln?Ser?Ala?Ala?Gly?Ser?Asn?Ser?Lys?Ser?Glu?Arg?Val?Ala?Ile
100?????????????????105?????????????????110
Gln?Glu?Glu?Val?Thr?Ala?Leu?Asn?Asp?Glu?Leu?Asn?Arg?Ile?Ala?Glu
115?????????????????120?????????????????125
Thr?Thr?Ser?Phe?Gly?Gly?Asn?Lys?Leu?Leu?Asn?Gly?Thr?Tyr?Gly?Thr
130?????????????????135?????????????????140
Lys?Ala?Met?Gln?Ile?Gly?Ala?Asp?Asn?Gly?Glu?Ala?Val?Met?Leu?Ser
145?????????????????150?????????????????155?????????????????160
Leu?Lys?Asp?Met?Arg?Ser?Asp?Asn?Val?Met?Met?Gly?Gly?Val?Ser?Tyr
165?????????????????170?????????????????175
Gln?Ala?Glu?Glu?Gly?Lys?Asp?Lys?Asn?Trp?Asn?Val?Ala?Ala?Gly?Asp
180?????????????????185?????????????????190
Asn?Asp?Leu?Thr?Ile?Ala?Leu?Thr?Asp?Ser?Phe?Gly?Asn?Glu?Gln?Glu
195?????????????????200?????????????????205
Ile?Glu?Ile?Asn?Ala?Lys?Ala?Gly?Asp?Asp?Ile?Glu?Glu?Leu?Ala?Thr
210?????????????????215?????????????????220
Tyr?Ile?Asn?Gly?Gln?Thr?Asp?Leu?Val?Lys?Ala?Ser?Val?Gly?Glu?Gly
225?????????????????230?????????????????235?????????????????240
Gly?Lys?Leu?Gln?Ile?Phe?Ala?Gly?Asn?Asn?Lys?Val?Gln?Gly?Glu?Ile
245?????????????????250?????????????????255
Ala?Phe?Ser?Gly?Ser?Leu?Ala?Gly?Glu?Leu?Gly?Leu?Gly?Glu?Gly?Lys
260?????????????????265?????????????????270
Asn?Val?Thr?Val?Asp?Thr?Ile?Asp?Val?Thr?Thr?Val?Gln?Gly?Ala?Gln
275?????????????????280?????????????????285
Glu?Ser?Val?Ala?Ile?Val?Asp?Ala?Ala?Leu?Lys?Tyr?Val?Asp?Ser?His
290?????????????????295?????????????????300
Arg?Ala?Glu?Leu?Gly?Ala?Phe?Gln?Asn?Arg?Phe?Asn?His?Ala?Ile?Ser
305?????????????????310?????????????????315?????????????????320
Asn?Leu?Asp?Asn?Ile?Asn?Glu?Asn?Val?Asn?Ala?Ser?Lys?Ser?Arg?Ile
325?????????????????330?????????????????335
Lys?Asp?Thr?Asp?Phe?Ala?Lys?Glu?Thr?Thr?Gln?Leu?Thr?Lys?Thr?Gln
340?????????????????345?????????????????350
Ile?Leu?Ser?Gln?Ala?Ser?Ser?Ser?Ile?Leu?Ala?Gln?Ala?Lys?Gln?Ala
355?????????????????360?????????????????365
Pro?Asn?Ser?Ala?Leu?Ser?Leu?Leu?Gly
370?????????????????375
<210>9
<211>1134
<212>DNA
＜213〉Vibrio vulnificus S103W FlaB gene
<400>9
atggcagtga?atgtaaatac?aaacgtagca?gcaatgacag?cacagcgtta?cctgaataac????60
gcaaacagcg?cacaacaaac?ttcgatggag?cgtctgtctt?caggtttcaa?aatcaacagt????120
gcaaaagatg?acgcagccgg?tctgcaaatc?tctaaccgct?tgaacgtaca?aagtcgcggt????180
ctagacgttg?cggtacgtaa?cgccaacgac?ggtatctcaa?tcgcacaaac?cgcagaaggt????240
gcgatgaacg?agaccaccaa?catcctacaa?cgtatgcgtg?acctatctct?acaatccgcg????300
aacggctgga?actcaaaatc?agagcgcgtg?gcgattcaag?aagaagtgac?agcattgaat????360
gacgagctaa?accgtattgc?agaaaccacg?tcttttggtg?gtaacaagct?gctaaacggt????420
acttacggca?cgaaagcaat?gcaaattggt?gcggataacg?gtgaagcggt?catgctttca????480
ctgaaagaca?tgcgctctga?caacgtgatg?atgggcggcg?tgagctacca?agctgaagaa????540
ggcaaagaca?agaactggaa?tgtggccgca?ggcgacaacg?acttgacgat?tgcactgaca????600
gacagctttg?gtaacgagca?agagatcgaa?atcaacgcga?aagcgggtga?tgacatcgaa????660
gagctagcga?cgtacatcaa?cggtcaaact?gaccttgtaa?aagcgtcagt?gggtgaaggc????720
ggcaagctac?agatctttgc?tggtaacaac?aaagttcaag?gtgaaattgc?tttctcaggt????780
agcctagctg?gtgaacttgg?cctaggcgaa?ggcaaaaacg?tcacggtaga?cacgattgac????840
gtgacaaccg?tacaaggtgc?gcaagagtcg?gtagcgattg?tggatgcggc?actgaaatac????900
gtagacagcc?accgtgcaga?gctgggtgca?ttccagaacc?gtttcaacca?tgcaatcagc????960
aacttggaca?acatcaacga?aaacgtgaac?gcgtcgaaga?gccgaatcaa?agataccgac????1020
ttcgcgaaag?aaacgactca?gttgaccaag?acacaaattc?tatcgcaagc?atcaagttcc????1080
attcttgcgc?aagcgaaaca?agcgccaaac?tcagcgctaa?gtctactagg?ctaa??????????1134
<210>10
<211>377
<212>PRT
＜213〉Vibrio vulnificus S103W FlaB amino acid
<400>10
Met?Ala?Val?Asn?Val?Asn?Thr?Asn?Val?Ala?Ala?Met?Thr?Ala?Gln?Arg
1???????????????5??????????????????10??????????????????15
Tyr?Leu?Asn?Asn?Ala?Asn?Ser?Ala?Gln?Gln?Thr?Ser?Met?Glu?Arg?Leu
20??????????????????25??????????????????30
Ser?Ser?Gly?Phe?Lys?Ile?Asn?Ser?Ala?Lys?Asp?Asp?Ala?Ala?Gly?Leu
35??????????????????40??????????????????45
Gln?Ile?Ser?Asn?Arg?Leu?Asn?Val?Gln?Ser?Arg?Gly?Leu?Asp?Val?Ala
50??????????????????55??????????????????60
Val?Arg?Asn?Ala?Asn?Asp?Gly?Ile?Ser?Ile?Ala?Gln?Thr?Ala?Glu?Gly
65??????????????????70??????????????????75??????????????????80
Ala?Met?Asn?Glu?Thr?Thr?Asn?Ile?Leu?Gln?Arg?Met?Arg?Asp?Leu?Ser
85??????????????????90??????????????????95
Leu?Gln?Ser?Ala?Asn?Gly?Trp?Asn?Ser?Lys?Ser?Glu?Arg?Val?Ala?Ile
100?????????????????105?????????????????110
Gln?Glu?Glu?Val?Thr?Ala?Leu?Asn?Asp?Glu?Leu?Asn?Arg?Ile?Ala?Glu
115?????????????????120?????????????????125
Thr?Thr?Ser?Phe?Gly?Gly?Asn?Lys?Leu?Leu?Asn?Gly?Thr?Tyr?Gly?Thr
130?????????????????135?????????????????140
Lys?Ala?Met?Gln?Ile?Gly?Ala?Asp?Asn?Gly?Glu?Ala?Val?Met?Leu?Ser
145?????????????????150?????????????????155?????????????????160
Leu?Lys?Asp?Met?Arg?Ser?Asp?Asn?Val?Met?Met?Gly?Gly?Val?Ser?Tyr
165?????????????????170?????????????????175
Gln?Ala?Glu?Glu?Gly?Lys?Asp?Lys?Asn?Trp?Asn?Val?Ala?Ala?Gly?Asp
180?????????????????185?????????????????190
Asn?Asp?Leu?Thr?Ile?Ala?Leu?Thr?Asp?Ser?Phe?Gly?Asn?Glu?Gln?Glu
195?????????????????200?????????????????205
Ile?Glu?Ile?Asn?Ala?Lys?Ala?Gly?Asp?Asp?Ile?Glu?Glu?Leu?Ala?Thr
210?????????????????215?????????????????220
Tyr?Ile?Asn?Gly?Gln?Thr?Asp?Leu?Val?Lys?Ala?Ser?Val?Gly?Glu?Gly
225?????????????????230?????????????????235?????????????????240
Gly?Lys?Leu?Gln?Ile?Phe?Ala?Gly?Asn?Asn?Lys?Val?Gln?Gly?Glu?Ile
245?????????????????250?????????????????255
Ala?Phe?Ser?Gly?Ser?Leu?Ala?Gly?Glu?Leu?Gly?Leu?Gly?Glu?Gly?Lys
260?????????????????265?????????????????270
Asn?Val?Thr?Val?Asp?Thr?Ile?Asp?Val?Thr?Thr?Val?Gln?Gly?Ala?Gln
275?????????????????280?????????????????285
Glu?Ser?Val?Ala?Ile?Val?Asp?Ala?Ala?Leu?Lys?Tyr?Val?Asp?Ser?His
290?????????????????295?????????????????300
Arg?Ala?Glu?Leu?Gly?Ala?Phe?Gln?Asn?Arg?Phe?Asn?His?Ala?Ile?Ser
305?????????????????310?????????????????315?????????????????320
Asn?Leu?Asp?Asn?Ile?Asn?Glu?Asn?Val?Asn?Ala?Ser?Lys?Ser?Arg?Ile
325?????????????????330?????????????????335
Lys?Asp?Thr?Asp?Phe?Ala?Lys?Glu?Thr?Thr?Gln?Leu?Thr?Lys?Thr?Gln
340?????????????????345?????????????????350
Ile?Leu?Ser?Gln?Ala?Ser?Ser?Ser?Ile?Leu?Ala?Gln?Ala?Lys?Gln?Ala
355?????????????????360?????????????????365
Pro?Asn?Ser?Ala?Leu?Ser?Leu?Leu?Gly
370?????????????????375
<210>11
<211>1134
<212>DNA
＜213〉Vibrio vulnificus E108A FlaB gene
<400>11
atggcagtga?atgtaaatac?aaacgtagca?gcaatgacag?cacagcgtta?cctgaataac????60
gcaaacagcg?cacaacaaac?ttcgatggag?cgtctgtctt?caggtttcaa?aatcaacagt????120
gcaaaagatg?acgcagccgg?tctgcaaatc?tctaaccgct?tgaacgtaca?aagtcgcggt????180
ctagacgttg?cggtacgtaa?cgccaacgac?ggtatctcaa?tcgcacaaac?cgcagaaggt????240
gcgatgaacg?agaccaccaa?catcctacaa?cgtatgcgtg?acctatctct?acaatccgcg????300
aacggctcaa?actcaaaatc?agcgcgcgtg?gcgattcaag?aagaagtgac?agcattgaat????360
gacgagctaa?accgtattgc?agaaaccacg?tcttttggtg?gtaacaagct?gctaaacggt????420
acttacggca?cgaaagcaat?gcaaattggt?gcggataacg?gtgaagcggt?catgctttca????480
ctgaaagaca?tgcgctctga?caacgtgatg?atgggcggcg?tgagctacca?agctgaagaa????540
ggcaaagaca?agaactggaa?tgtggccgca?ggcgacaacg?acttgacgat?tgcactgaca????600
gacagctttg?gtaacgagca?agagatcgaa?atcaacgcga?aagcgggtga?tgacatcgaa????660
gagctagcga?cgtacatcaa?cggtcaaact?gaccttgtaa?aagcgtcagt?gggtgaaggc????720
ggcaagctac?agatctttgc?tggtaacaac?aaagttcaag?gtgaaattgc?tttctcaggt????780
agcctagctg?gtgaacttgg?cctaggcgaa?ggcaaaaacg?tcacggtaga?cacgattgac????840
gtgacaaccg?tacaaggtgc?gcaagagtcg?gtagcgattg?tggatgcggc?actgaaatac????900
gtagacagcc?accgtgcaga?gctgggtgca?ttccagaacc?gtttcaacca?tgcaatcagc????960
aacttggaca?acatcaacga?aaacgtgaac?gcgtcgaaga?gccgaatcaa?agataccgac????1020
ttcgcgaaag?aaacgactca?gttgaccaag?acacaaattc?tatcgcaagc?atcaagttcc????1080
attcttgcgc?aagcgaaaca?agcgccaaac?tcagcgctaa?gtctactagg?ctaa??????????1134
<210>12
<211>377
<212>PRT
＜213〉Vibrio vulnificus E108A FlaB amino acid
<400>12
Met?Ala?Val?Asn?Val?Asn?Thr?Asn?Val?Ala?Ala?Met?Thr?Ala?Gln?Arg
1???????????????5??????????????????10??????????????????15
Tyr?Leu?Asn?Asn?Ala?Asn?Ser?Ala?Gln?Gln?Thr?Ser?Met?Glu?Arg?Leu
20??????????????????25??????????????????30
Ser?Ser?Gly?Phe?Lys?Ile?Asn?Ser?Ala?Lys?Asp?Asp?Ala?Ala?Gly?Leu
35??????????????????40??????????????????45
Gln?Ile?Ser?Asn?Arg?Leu?Asn?Val?Gln?Ser?Arg?Gly?Leu?Asp?Val?Ala
50??????????????????55??????????????????60
Val?Arg?Asn?Ala?Asn?Asp?Gly?Ile?Ser?Ile?Ala?Gln?Thr?Ala?Glu?Gly
65??????????????????70??????????????????75??????????????????80
Ala?Met?Asn?Glu?Thr?Thr?Asn?Ile?Leu?Gln?Arg?Met?Arg?Asp?Leu?Ser
85??????????????????90??????????????????95
Leu?Gln?Ser?Ala?Asn?Gly?Ser?Asn?Ser?Lys?Ser?Ala?Arg?Val?Ala?Ile
100?????????????????105?????????????????110
Gln?Glu?Glu?Val?Thr?Ala?Leu?Asn?Asp?Glu?Leu?Asn?Arg?Ile?Ala?Glu
115?????????????????120?????????????????125
Thr?Thr?Ser?Phe?Gly?Gly?Asn?Lys?Leu?Leu?Asn?Gly?Thr?Tyr?Gly?Thr
130?????????????????135?????????????????140
Lys?Ala?Met?Gln?Ile?Gly?Ala?Asp?Asn?Gly?Glu?Ala?Val?Met?Leu?Ser
145?????????????????150?????????????????155?????????????????160
Leu?Lys?Asp?Met?Arg?Ser?Asp?Asn?Val?Met?Met?Gly?Gly?Val?Ser?Tyr
165?????????????????170?????????????????175
Gln?Ala?Glu?Glu?Gly?Lys?Asp?Lys?Asn?Trp?Asn?Val?Ala?Ala?Gly?Asp
180?????????????????185?????????????????190
Asn?Asp?Leu?Thr?Ile?Ala?Leu?Thr?Asp?Ser?Phe?Gly?Asn?Glu?Gln?Glu
195?????????????????200?????????????????205
Ile?Glu?Ile?Asn?Ala?Lys?Ala?Gly?Asp?Asp?Ile?Glu?Glu?Leu?Ala?Thr
210?????????????????215?????????????????220
Tyr?Ile?Asn?Gly?Gln?Thr?Asp?Leu?Val?Lys?Ala?Ser?Val?Gly?Glu?Gly
225?????????????????230?????????????????235?????????????????240
Gly?Lys?Leu?Gln?Ile?Phe?Ala?Gly?Asn?Asn?Lys?Val?Gln?Gly?Glu?Ile
245?????????????????250?????????????????255
Ala?Phe?Ser?Gly?Ser?Leu?Ala?Gly?Glu?Leu?Gly?Leu?Gly?Glu?Gly?Lys
260?????????????????265?????????????????270
Asn?Val?Thr?Val?Asp?Thr?Ile?Asp?Val?Thr?Thr?Val?Gln?Gly?Ala?Gln
275?????????????????280?????????????????285
Glu?Ser?Val?Ala?Ile?Val?Asp?Ala?Ala?Leu?Lys?Tyr?Val?Asp?Ser?His
290?????????????????295?????????????????300
Arg?Ala?Glu?Leu?Gly?Ala?Phe?Gln?Asn?Arg?Phe?Asn?His?Ala?Ile?Ser
305?????????????????310?????????????????315?????????????????320
Asn?Leu?Asp?Asn?Ile?Asn?Glu?Asn?Val?Asn?Ala?Ser?Lys?Ser?Arg?Ile
325?????????????????330?????????????????335
Lys?Asp?Thr?Asp?Phe?Ala?Lys?Glu?Thr?Thr?Gln?Leu?Thr?Lys?Thr?Gln
340?????????????????345?????????????????350
Ile?Leu?Ser?Gln?Ala?Ser?Ser?Ser?Ile?Leu?Ala?Gln?Ala?Lys?Gln?Ala
355?????????????????360?????????????????365
Pro?Asn?Ser?Ala?Leu?Ser?Leu?Leu?Gly
370?????????????????375
<210>13
<211>1134
<212>DNA
＜213〉Vibrio vulnificus N135D FlaB gene
<400>13
atggcagtga?atgtaaatac?aaacgtagca?gcaatgacag?cacagcgtta?cctgaataac????60
gcaaacagcg?cacaacaaac?ttcgatggag?cgtctgtctt?caggtttcaa?aatcaacagt????120
gcaaaagatg?acgcagccgg?tctgcaaatc?tctaaccgct?tgaacgtaca?aagtcgcggt????180
ctagacgttg?cggtacgtaa?cgccaacgac?ggtatctcaa?tcgcacaaac?cgcagaaggt????240
gcgatgaacg?agaccaccaa?catcctacaa?cgtatgcgtg?acctatctct?acaatccgcg????300
aacggctcaa?actcaaaatc?agagcgcgtg?gcgattcaag?aagaagtgac?agcattgaat????360
gacgagctaa?accgtattgc?agaaaccacg?tcttttggtg?gtgacaagct?gctaaacggt????420
acttacggca?cgaaagcaat?gcaaattggt?gcggataacg?gtgaagcggt?catgctttca????480
ctgaaagaca?tgcgctctga?caacgtgatg?atgggcggcg?tgagctacca?agctgaagaa????540
ggcaaagaca?agaactggaa?tgtggccgca?ggcgacaacg?acttgacgat?tgcactgaca????600
gacagctttg?gtaacgagca?agagatcgaa?atcaacgcga?aagcgggtga?tgacatcgaa????660
gagctagcga?cgtacatcaa?cggtcaaact?gaccttgtaa?aagcgtcagt?gggtgaaggc????720
ggcaagctac?agatctttgc?tggtaacaac?aaagttcaag?gtgaaattgc?tttctcaggt????780
agcctagctg?gtgaacttgg?cctaggcgaa?ggcaaaaacg?tcacggtaga?cacgattgac????840
gtgacaaccg?tacaaggtgc?gcaagagtcg?gtagcgattg?tggatgcggc?actgaaatac????900
gtagacagcc?accgtgcaga?gctgggtgca?ttccagaacc?gtttcaacca?tgcaatcagc????960
aacttggaca?acatcaacga?aaacgtgaac?gcgtcgaaga?gccgaatcaa?agataccgac????1020
ttcgcgaaag?aaacgactca?gttgaccaag?acacaaattc?tatcgcaagc?atcaagttcc????1080
attcttgcgc?aagcgaaaca?agcgccaaac?tcagcgctaa?gtctactagg?ctaa??????????1134
<210>14
<211>377
<212>PRT
＜213〉Vibrio vulnificus N135D FlaB amino acid
<400>14
Met?Ala?Val?Asn?Val?Asn?Thr?Asn?Val?Ala?Ala?Met?Thr?Ala?Gln?Arg
1???????????????5??????????????????10??????????????????15
Tyr?Leu?Asn?Asn?Ala?Asn?Ser?Ala?Gln?Gln?Thr?Ser?Met?Glu?Arg?Leu
20??????????????????25??????????????????30
Ser?Ser?Gly?Phe?Lys?Ile?Asn?Ser?Ala?Lys?Asp?Asp?Ala?Ala?Gly?Leu
35??????????????????40??????????????????45
Gln?Ile?Ser?Asn?Arg?Leu?Asn?Val?Gln?Ser?Arg?Gly?Leu?Asp?Val?Ala
50??????????????????55??????????????????60
Val?Arg?Asn?Ala?Asn?Asp?Gly?Ile?Ser?Ile?Ala?Gln?Thr?Ala?Glu?Gly
65??????????????????70??????????????????75??????????????????80
Ala?Met?Asn?Glu?Thr?Thr?Asn?Ile?Leu?Gln?Arg?Met?Arg?Asp?Leu?Ser
85??????????????????90??????????????????95
Leu?Gln?Ser?Ala?Asn?Gly?Ser?Asn?Ser?Lys?Ser?Glu?Arg?Val?Ala?Ile
100?????????????????105?????????????????110
Gln?Glu?Glu?Val?Thr?Ala?Leu?Asn?Asp?Glu?Leu?Asn?Arg?Ile?Ala?Glu
115?????????????????120?????????????????125
Thr?Thr?Ser?Phe?Gly?Gly?Asp?Lys?Leu?Leu?Asn?Gly?Thr?Tyr?Gly?Thr
130?????????????????135?????????????????140
Lys?Ala?Met?Gln?Ile?Gly?Ala?Asp?Asn?Gly?Glu?Ala?Val?Met?Leu?Ser
145?????????????????150?????????????????155?????????????????160
Leu?Lys?Asp?Met?Arg?Ser?Asp?Asn?Val?Met?Met?Gly?Gly?Val?Ser?Tyr
165?????????????????170?????????????????175
Gln?Ala?Glu?Glu?Gly?Lys?Asp?Lys?Asn?Trp?Asn?Val?Ala?Ala?Gly?Asp
180?????????????????185?????????????????190
Asn?Asp?Leu?Thr?Ile?Ala?Leu?Thr?Asp?Ser?Phe?Gly?Asn?Glu?Gln?Glu
195?????????????????200?????????????????205
Ile?Glu?Ile?Asn?Ala?Lys?Ala?Gly?Asp?Asp?Ile?Glu?Glu?Leu?Ala?Thr
210?????????????????215?????????????????220
Tyr?Ile?Asn?Gly?Gln?Thr?Asp?Leu?Val?Lys?Ala?Ser?Val?Gly?Glu?Gly
225?????????????????230?????????????????235?????????????????240
Gly?Lys?Leu?Gln?Ile?Phe?Ala?Gly?Asn?Asn?Lys?Val?Gln?Gly?Glu?Ile
245?????????????????250?????????????????255
Ala?Phe?Ser?Gly?Ser?Leu?Ala?Gly?Glu?Leu?Gly?Leu?Gly?Glu?Gly?Lys
260?????????????????265?????????????????270
Asn?Val?Thr?Val?Asp?Thr?Ile?Asp?Val?Thr?Thr?Val?Gln?Gly?Ala?Gln
275?????????????????280?????????????????285
Glu?Ser?Val?Ala?Ile?Val?Asp?Ala?Ala?Leu?Lys?Tyr?Val?Asp?Ser?His
290?????????????????295?????????????????300
Arg?Ala?Glu?Leu?Gly?Ala?Phe?Gln?Asn?Arg?Phe?Asn?His?Ala?Ile?Ser
305?????????????????310?????????????????315?????????????????320
Asn?Leu?Asp?Asn?Ile?Asn?Glu?Asn?Val?Asn?Ala?Ser?Lys?Ser?Arg?Ile
325?????????????????330?????????????????335
Lys?Asp?Thr?Asp?Phe?Ala?Lys?Glu?Thr?Thr?Gln?Leu?Thr?Lys?Thr?Gln
340?????????????????345?????????????????350
Ile?Leu?Ser?Gln?Ala?Ser?Ser?Ser?Ile?Leu?Ala?Gln?Ala?Lys?Gln?Ala
355?????????????????360?????????????????365
Pro?Asn?Ser?Ala?Leu?Ser?Leu?Leu?Gly
370?????????????????375
<210>15
<211>1134
<212>DNA
＜213〉Vibrio vulnificus A151W FlaB gene
<400>15
atggcagtga?atgtaaatac?aaacgtagca?gcaatgacag?cacagcgtta?cctgaataac????60
gcaaacagcg?cacaacaaac?ttcgatggag?cgtctgtctt?caggtttcaa?aatcaacagt????120
gcaaaagatg?acgcagccgg?tctgcaaatc?tctaaccgct?tgaacgtaca?aagtcgcggt????180
ctagacgttg?cggtacgtaa?cgccaacgac?ggtatctcaa?tcgcacaaac?cgcagaaggt????240
gcgatgaacg?agaccaccaa?catcctacaa?cgtatgcgtg?acctatctct?acaatccgcg????300
aacggctcaa?actcaaaatc?agagcgcgtg?gcgattcaag?aagaagtgac?agcattgaat????360
gacgagctaa?accgtattgc?agaaaccacg?tcttttggtg?gtaacaagct?gctaaacggt????420
acttacggca?cgaaagcaat?gcaaattggt?tgggataacg?gtgaagcggt?catgctttca????480
ctgaaagaca?tgcgctctga?caacgtgatg?atgggcggcg?tgagctacca?agctgaagaa????540
ggcaaagaca?agaactggaa?tgtggccgca?ggcgacaacg?acttgacgat?tgcactgaca????600
gacagctttg?gtaacgagca?agagatcgaa?atcaacgcga?aagcgggtga?tgacatcgaa????660
gagctagcga?cgtacatcaa?cggtcaaact?gaccttgtaa?aagcgtcagt?gggtgaaggc????720
ggcaagctac?agatctttgc?tggtaacaac?aaagttcaag?gtgaaattgc?tttctcaggt????780
agcctagctg?gtgaacttgg?cctaggcgaa?ggcaaaaacg?tcacggtaga?cacgattgac????840
gtgacaaccg?tacaaggtgc?gcaagagtcg?gtagcgattg?tggatgcggc?actgaaatac????900
gtagacagcc?accgtgcaga?gctgggtgca?ttccagaacc?gtttcaacca?tgcaatcagc????960
aacttggaca?acatcaacga?aaacgtgaac?gcgtcgaaga?gccgaatcaa?agataccgac????1020
ttcgcgaaag?aaacgactca?gttgaccaag?acacaaattc?tatcgcaagc?atcaagttcc????1080
attcttgcgc?aagcgaaaca?agcgccaaac?tcagcgctaa?gtctactagg?ctaa??????????1134
<210>16
<211>377
<212>PRT
＜213〉Vibrio vulnificus A151W FlaB amino acid
<400>16
Met?Ala?Val?Asn?Val?Asn?Thr?Asn?Val?Ala?Ala?Met?Thr?Ala?Gln?Arg
1???????????????5??????????????????10??????????????????15
Tyr?Leu?Asn?Asn?Ala?Asn?Ser?Ala?Gln?Gln?Thr?Ser?Met?Glu?Arg?Leu
20??????????????????25??????????????????30
Ser?Ser?Gly?Phe?Lys?Ile?Asn?Ser?Ala?Lys?Asp?Asp?Ala?Ala?Gly?Leu
35??????????????????40??????????????????45
Gln?Ile?Ser?Asn?Arg?Leu?Asn?Val?Gln?Ser?Arg?Gly?Leu?Asp?Val?Ala
50??????????????????55??????????????????60
Val?Arg?Asn?Ala?Asn?Asp?Gly?Ile?Ser?Ile?Ala?Gln?Thr?Ala?Glu?Gly
65??????????????????70??????????????????75??????????????????80
Ala?Met?Asn?Glu?Thr?Thr?Asn?Ile?Leu?Gln?Arg?Met?Arg?Asp?Leu?Ser
85??????????????????90??????????????????95
Leu?Gln?Ser?Ala?Asn?Gly?Ser?Asn?Ser?Lys?Ser?Glu?Arg?Val?Ala?Ile
100?????????????????105?????????????????110
Gln?Glu?Glu?Val?Thr?Ala?Leu?Asn?Asp?Glu?Leu?Asn?Arg?Ile?Ala?Glu
115?????????????????120?????????????????125
Thr?Thr?Ser?Phe?Gly?Gly?Asn?Lys?Leu?Leu?Asn?Gly?Thr?Tyr?Gly?Thr
130?????????????????135?????????????????140
Lys?Ala?Met?Gln?Ile?Gly?Trp?Asp?Asn?Gly?Glu?Ala?Val?Met?Leu?Ser
145?????????????????150?????????????????155?????????????????160
Leu?Lys?Asp?Met?Arg?Ser?Asp?Asn?Val?Met?Met?Gly?Gly?Val?Ser?Tyr
165?????????????????170?????????????????175
Gln?Ala?Glu?Glu?Gly?Lys?Asp?Lys?Asn?Trp?Asn?Val?Ala?Ala?Gly?Asp
180?????????????????185?????????????????190
Asn?Asp?Leu?Thr?Ile?Ala?Leu?Thr?Asp?Ser?Phe?Gly?Asn?Glu?Gln?Glu
195?????????????????200?????????????????205
Ile?Glu?Ile?Asn?Ala?Lys?Ala?Gly?Asp?Asp?Ile?Glu?Glu?Leu?Ala?Thr
210?????????????????215?????????????????220
Tyr?Ile?Asn?Gly?Gln?Thr?Asp?Leu?Val?Lys?Ala?Ser?Val?Gly?Glu?Gly
225?????????????????230?????????????????235?????????????????240
Gly?Lys?Leu?Gln?Ile?Phe?Ala?Gly?Asn?Asn?Lys?Val?Gln?Gly?Glu?Ile
245?????????????????250?????????????????255
Ala?Phe?Ser?Gly?Ser?Leu?Ala?Gly?Glu?Leu?Gly?Leu?Gly?Glu?Gly?Lys
260?????????????????265?????????????????270
Asn?Val?Thr?Val?Asp?Thr?Ile?Asp?Val?Thr?Thr?Val?Gln?Gly?Ala?Gln
275?????????????????280?????????????????285
Glu?Ser?Val?Ala?Ile?Val?Asp?Ala?Ala?Leu?Lys?Tyr?Val?Asp?Ser?His
290?????????????????295?????????????????300
Arg?Ala?Glu?Leu?Gly?Ala?Phe?Gln?Asn?Arg?Phe?Asn?His?Ala?Ile?Ser
305?????????????????310?????????????????315?????????????????320
Asn?Leu?Asp?Asn?Ile?Asn?Glu?Asn?Val?Asn?Ala?Ser?Lys?Ser?Arg?Ile
325?????????????????330?????????????????335
Lys?Asp?Thr?Asp?Phe?Ala?Lys?Glu?Thr?Thr?Gln?Leu?Thr?Lys?Thr?Gln
340?????????????????345?????????????????350
Ile?Leu?Ser?Gln?Ala?Ser?Ser?Ser?Ile?Leu?Ala?Gln?Ala?Lys?Gln?Ala
355?????????????????360?????????????????365
Pro?Asn?Ser?Ala?Leu?Ser?Leu?Leu?Gly
370?????????????????375
<210>17
<211>1134
<212>DNA
＜213〉Vibrio vulnificus N153W FlaB gene
<400>17
atggcagtga?atgtaaatac?aaacgtagca?gcaatgacag?cacagcgtta?cctgaataac????60
gcaaacagcg?cacaacaaac?ttcgatggag?cgtctgtctt?caggtttcaa?aatcaacagt????120
gcaaaagatg?acgcagccgg?tctgcaaatc?tctaaccgct?tgaacgtaca?aagtcgcggt????180
ctagacgttg?cggtacgtaa?cgccaacgac?ggtatctcaa?tcgcacaaac?cgcagaaggt????240
gcgatgaacg?agaccaccaa?catcctacaa?cgtatgcgtg?acctatctct?acaatccgcg????300
aacggctcaa?actcaaaatc?agagcgcgtg?gcgattcaag?aagaagtgac?agcattgaat????360
gacgagctaa?accgtattgc?agaaaccacg?tcttttggtg?gtaacaagct?gctaaacggt????420
acttacggca?cgaaagcaat?gcaaattggt?gcggattggg?gtgaagcggt?catgctttca????480
ctgaaagaca?tgcgctctga?caacgtgatg?atgggcggcg?tgagctacca?agctgaagaa????540
ggcaaagaca?agaactggaa?tgtggccgca?ggcgacaacg?acttgacgat?tgcactgaca????600
gacagctttg?gtaacgagca?agagatcgaa?atcaacgcga?aagcgggtga?tgacatcgaa????660
gagctagcga?cgtacatcaa?cggtcaaact?gaccttgtaa?aagcgtcagt?gggtgaaggc????720
ggcaagctac?agatctttgc?tggtaacaac?aaagttcaag?gtgaaattgc?tttctcaggt????780
agcctagctg?gtgaacttgg?cctaggcgaa?ggcaaaaacg?tcacggtaga?cacgattgac????840
gtgacaaccg?tacaaggtgc?gcaagagtcg?gtagcgattg?tggatgcggc?actgaaatac????900
gtagacagcc?accgtgcaga?gctgggtgca?ttccagaacc?gtttcaacca?tgcaatcagc????960
aacttggaca?acatcaacga?aaacgtgaac?gcgtcgaaga?gccgaatcaa?agataccgac????1020
ttcgcgaaag?aaacgactca?gttgaccaag?acacaaattc?tatcgcaagc?atcaagttcc????1080
attcttgcgc?aagcgaaaca?agcgccaaac?tcagcgctaa?gtctactagg?ctaa??????????1134
<210>18
<211>377
<212>PRT
＜213〉Vibrio vulnificus N153W FlaB amino acid
<400>18
Met?Ala?Val?Asn?Val?Asn?Thr?Asn?Val?Ala?Ala?Met?Thr?Ala?Gln?Arg
1???????????????5??????????????????10??????????????????15
Tyr?Leu?Asn?Asn?Ala?Asn?Ser?Ala?Gln?Gln?Thr?Ser?Met?Glu?Arg?Leu
20??????????????????25??????????????????30
Ser?Ser?Gly?Phe?Lys?Ile?Asn?Ser?Ala?Lys?Asp?Asp?Ala?Ala?Gly?Leu
35??????????????????40??????????????????45
Gln?Ile?Ser?Asn?Arg?Leu?Asn?Val?Gln?Ser?Arg?Gly?Leu?Asp?Val?Ala
50??????????????????55??????????????????60
Val?Arg?Asn?Ala?Asn?Asp?Gly?Ile?Ser?Ile?Ala?Gln?Thr?Ala?Glu?Gly
65??????????????????70??????????????????75??????????????????80
Ala?Met?Asn?Glu?Thr?Thr?Asn?Ile?Leu?Gln?Arg?Met?Arg?Asp?Leu?Ser
85??????????????????90??????????????????95
Leu?Gln?Ser?Ala?Asn?Gly?Ser?Asn?Ser?Lys?Ser?Glu?Arg?Val?Ala?Ile
100?????????????????105?????????????????110
Gln?Glu?Glu?Val?Thr?Ala?Leu?Asn?Asp?Glu?Leu?Asn?Arg?Ile?Ala?Glu
115?????????????????120?????????????????125
Thr?Thr?Ser?Phe?Gly?Gly?Asn?Lys?Leu?Leu?Asn?Gly?Thr?Tyr?Gly?Thr
130?????????????????135?????????????????140
Lys?Ala?Met?Gln?Ile?Gly?Ala?Asp?Trp?Gly?Glu?Ala?Val?Met?Leu?Ser
145?????????????????150?????????????????155?????????????????160
Leu?Lys?Asp?Met?Arg?Ser?Asp?Asn?Val?Met?Met?Gly?Gly?Val?Ser?Tyr
165?????????????????170?????????????????175
Gln?Ala?Glu?Glu?Gly?Lys?Asp?Lys?Asn?Trp?Asn?Val?Ala?Ala?Gly?Asp
180?????????????????185?????????????????190
Asn?Asp?Leu?Thr?Ile?Ala?Leu?Thr?Asp?Ser?Phe?Gly?Asn?Glu?Gln?Glu
195?????????????????200?????????????????205
Ile?Glu?Ile?Asn?Ala?Lys?Ala?Gly?Asp?Asp?Ile?Glu?Glu?Leu?Ala?Thr
210?????????????????215?????????????????220
Tyr?Ile?Asn?Gly?Gln?Thr?Asp?Leu?Val?Lys?Ala?Ser?Val?Gly?Glu?Gly
225?????????????????230?????????????????235?????????????????240
Gly?Lys?Leu?Gln?Ile?Phe?Ala?Gly?Asn?Asn?Lys?Val?Gln?Gly?Glu?Ile
245?????????????????250?????????????????255
Ala?Phe?Ser?Gly?Ser?Leu?Ala?Gly?Glu?Leu?Gly?Leu?Gly?Glu?Gly?Lys
260?????????????????265?????????????????270
Asn?Val?Thr?Val?Asp?Thr?Ile?Asp?Val?Thr?Thr?Val?Gln?Gly?Ala?Gln
275?????????????????280?????????????????285
Glu?Ser?Val?Ala?Ile?Val?Asp?Ala?Ala?Leu?Lys?Tyr?Val?Asp?Ser?His
290?????????????????295?????????????????300
Arg?Ala?Glu?Leu?Gly?Ala?Phe?Gln?Asn?Arg?Phe?Asn?His?Ala?Ile?Ser
305?????????????????310?????????????????315?????????????????320
Asn?Leu?Asp?Asn?Ile?Asn?Glu?Asn?Val?Asn?Ala?Ser?Lys?Ser?Arg?Ile
325?????????????????330?????????????????335
Lys?Asp?Thr?Asp?Phe?Ala?Lys?Glu?Thr?Thr?Gln?Leu?Thr?Lys?Thr?Gln
340?????????????????345?????????????????350
Ile?Leu?Ser?Gln?Ala?Ser?Ser?Ser?Ile?Leu?Ala?Gln?Ala?Lys?Gln?Ala
355?????????????????360?????????????????365
Pro?Asn?Ser?Ala?Leu?Ser?Leu?Leu?Gly
370?????????????????375
<210>19
<211>1134
<212>DNA
＜213〉Vibrio vulnificus V291W FlaB gene
<400>19
atggcagtga?atgtaaatac?aaacgtagca?gcaatgacag?cacagcgtta?cctgaataac????60
gcaaacagcg?cacaacaaac?ttcgatggag?cgtctgtctt?caggtttcaa?aatcaacagt????120
gcaaaagatg?acgcagccgg?tctgcaaatc?tctaaccgct?tgaacgtaca?aagtcgcggt????180
ctagacgttg?cggtacgtaa?cgccaacgac?ggtatctcaa?tcgcacaaac?cgcagaaggt????240
gcgatgaacg?agaccaccaa?catcctacaa?cgtatgcgtg?acctatctct?acaatccgcg????300
aacggctcaa?actcaaaatc?agagcgcgtg?gcgattcaag?aagaagtgac?agcattgaat????360
gacgagctaa?accgtattgc?agaaaccacg?tcttttggtg?gtaacaagct?gctaaacggt????420
acttacggca?cgaaagcaat?gcaaattggt?gcggataacg?gtgaagcggt?catgctttca????480
ctgaaagaca?tgcgctctga?caacgtgatg?atgggcggcg?tgagctacca?agctgaagaa????540
ggcaaagaca?agaactggaa?tgtggccgca?ggcgacaacg?acttgacgat?tgcactgaca????600
gacagctttg?gtaacgagca?agagatcgaa?atcaacgcga?aagcgggtga?tgacatcgaa????660
gagctagcga?cgtacatcaa?cggtcaaact?gaccttgtaa?aagcgtcagt?gggtgaaggc????720
ggcaagctac?agatctttgc?tggtaacaac?aaagttcaag?gtgaaattgc?tttctcaggt????780
agcctagctg?gtgaacttgg?cctaggcgaa?ggcaaaaacg?tcacggtaga?cacgattgac????840
gtgacaaccg?tacaaggtgc?gcaagagtcg?tgggcgattg?tggatgcggc?actgaaatac????900
gtagacagcc?accgtgcaga?gctgggtgca?ttccagaacc?gtttcaacca?tgcaatcagc????960
aacttggacaacatcaacga?aaacgtgaac?gcgtcgaaga?gccgaatcaa?agataccgac????1020
ttcgcgaaag?aaacgactca?gttgaccaag?acacaaattc?tatcgcaagc?atcaagttcc???1080
attcttgcgc?aagcgaaaca?agcgccaaac?tcagcgctaa?gtctactagg?ctaa?????????1134
<210>20
<211>377
<212>PRT
＜213〉Vibrio vulnificus V291W FlaB amino acid
<400>20
Met?Ala?Val?Asn?Val?Asn?Thr?Asn?Val?Ala?Ala?Met?Thr?Ala?Gln?Arg
1???????????????5??????????????????10??????????????????15
Tyr?Leu?Asn?Asn?Ala?Asn?Ser?Ala?Gln?Gln?Thr?Ser?Met?Glu?Arg?Leu
20??????????????????25??????????????????30
Ser?Ser?Gly?Phe?Lys?Ile?Asn?Ser?Ala?Lys?Asp?Asp?Ala?Ala?Gly?Leu
35??????????????????40??????????????????45
Gln?Ile?Ser?Asn?Arg?Leu?Asn?Val?Gln?Ser?Arg?Gly?Leu?Asp?Val?Ala
50??????????????????55??????????????????60
Val?Arg?Asn?Ala?Asn?Asp?Gly?Ile?Ser?Ile?Ala?Gln?Thr?Ala?Glu?Gly
65??????????????????70??????????????????75??????????????????80
Ala?Met?Asn?Glu?Thr?Thr?Asn?Ile?Leu?Gln?Arg?Met?Arg?Asp?Leu?Ser
85??????????????????90??????????????????95
Leu?Gln?Ser?Ala?Asn?Gly?Ser?Asn?Ser?Lys?Ser?Glu?Arg?Val?Ala?Ile
100?????????????????105?????????????????110
Gln?Glu?Glu?Val?Thr?Ala?Leu?Asn?Asp?Glu?Leu?Asn?Arg?Ile?Ala?Glu
115?????????????????120?????????????????125
Thr?Thr?Ser?Phe?Gly?Gly?Asn?Lys?Leu?Leu?Asn?Gly?Thr?Tyr?Gly?Thr
130?????????????????135?????????????????140
Lys?Ala?Met?Gln?Ile?Gly?Ala?Asp?Asn?Gly?Glu?Ala?Val?Met?Leu?Ser
145?????????????????150?????????????????155?????????????????160
Leu?Lys?Asp?Met?Arg?Ser?Asp?Asn?Val?Met?Met?Gly?Gly?Val?Ser?Tyr
165?????????????????170?????????????????175
Gln?Ala?Glu?Glu?Gly?Lys?Asp?Lys?Asn?Trp?Asn?Val?Ala?Ala?Gly?Asp
180?????????????????185?????????????????190
Asn?Asp?Leu?Thr?Ile?Ala?Leu?Thr?Asp?Ser?Phe?Gly?Asn?Glu?Gln?Glu
195?????????????????200?????????????????205
Ile?Glu?Ile?Asn?Ala?Lys?Ala?Gly?Asp?Asp?Ile?Glu?Glu?Leu?Ala?Thr
210?????????????????215?????????????????220
Tyr?Ile?Asn?Gly?Gln?Thr?Asp?Leu?Val?Lys?Ala?Ser?Val?Gly?Glu?Gly
225?????????????????230?????????????????235?????????????????240
Gly?Lys?Leu?Gln?Ile?Phe?Ala?Gly?Asn?Asn?Lys?Val?Gln?Gly?Glu?Ile
245?????????????????250?????????????????255
Ala?Phe?Ser?Gly?Ser?Leu?Ala?Gly?Glu?Leu?Gly?Leu?Gly?Glu?Gly?Lys
260?????????????????265?????????????????270
Asn?Val?Thr?Val?Asp?Thr?Ile?Asp?Val?Thr?Thr?Val?Gln?Gly?Ala?Gln
275?????????????????280?????????????????285
Glu?Ser?Trp?Ala?Ile?Val?Asp?Ala?Ala?Leu?Lys?Tyr?Val?Asp?Ser?His
290?????????????????295?????????????????300
Arg?Ala?Glu?Leu?Gly?Ala?Phe?Gln?Asn?Arg?Phe?Asn?His?Ala?Ile?Ser
305?????????????????310?????????????????315?????????????????320
Asn?Leu?Asp?Asn?Ile?Asn?Glu?Asn?Val?Asn?Ala?Ser?Lys?Ser?Arg?Ile
325?????????????????330?????????????????335
Lys?Asp?Thr?Asp?Phe?Ala?Lys?Glu?Thr?Thr?Gln?Leu?Thr?Lys?Thr?Gln
340?????????????????345?????????????????350
Ile?Leu?Ser?Gln?Ala?Ser?Ser?Ser?Ile?Leu?Ala?Gln?Ala?Lys?Gln?Ala
355?????????????????360?????????????????365
Pro?Asn?Ser?Ala?Leu?Ser?Leu?Leu?Gly
370?????????????????375
<210>21
<211>38
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-V5 primer
<400>21
gcggccgcat?ggcagtgaat?gtaaatgtaa?atacaaac????38
<210>22
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-V4 primer
<400>22
cccggggcct?agtagactta?gcgctga????????????27
<210>23
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD1 primer
<400>23
gtacgtaacg?ccaacgcagg?tatctcaatc??????????30
<210>24
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD2 primer
<400>24
gattgagata?cctgcgttgg?cgttacgtac???????????30
<210>25
<211>36
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD13-2 primer
<400>25
catcctacaa?cgtatggctg?acctatctct?acaatc????36
<210>26
<211>36
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD14-2 primer
<400>26
gattgtagag?ataggtcagc?catacgttgt?aggatg????36
<210>27
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD17 primer
<400>27
gcgtgaccta?tcttggcaat?ccgcgaacgg???????????30
<210>28
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD18 primer
<400>28
ccgttcgcgg?attgccaaga?taggtcacgc???????????30
<210>29
<211>32
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD9-3 primer
<400>29
ctacaatccg?cggccggctc?aaactcaaaa?tc????????32
<210>30
<211>32
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD10-3 primer
<400>30
gattttgagt?ttgagccggc?cgcggattgt?ag????32
<210>31
<211>31
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD15 primer
<400>31
caatccgcga?acggctggaa?ctcaaaatca?g?????31
<210>32
<211>31
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD16 primer
<400>32
ctgattttga?gttccagccg?ttcgcggatt?g?????31
<210>33
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD11 primer
<400>33
caaactcaaa?atcagcgcgc?gtggcgattc???????30
<210>34
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD12 primer
<400>34
gaatcgccac?gcgcgctgat?tttgagtttg?????30
<210>35
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD3 primer
<400>35
cgtcttttgg?tggtgacaag?ctgctaaacg?????30
<210>36
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD4 primer
<400>36
cgtttagcag?cttgtcacca?ccaaaagacg??????30
<210>37
<211>33
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD5 primer
<400>37
gcaatgcaaa?ttggttggga?taacggtgaa?gcg????33
<210>38
<211>33
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD6 primer
<400>38
cgcttcaccg?ttatcccaac?caatttgcat?tgc????33
<210>39
<211>33
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD7 primer
<400>39
caaattggtg?cggattgggg?tgaagcggtc?atg????33
<210>40
<211>33
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD8 primer
<400>40
catgaccgct?tcaccccaat?ccgcaccaat?ttg????33
<210>41
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD19 primer
<400>41
gcgcaagagt?cgtgggcgat?tgtggatgcg????????30
<210>42
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉FlaB-SD20 primer
<400>42
cgcatccaca?atcgcccacg?actcttgcgc?????????????????????????????????????30
<210>43
<211>1134
<212>DNA
＜213〉Vibrio vulnificus D70A/N135D FlaB gene
<400>43
atggcagtga?atgtaaatac?aaacgtagca?gcaatgacag?cacagcgtta?cctgaataac????60
gcaaacagcg?cacaacaaac?ttcgatggag?cgtctgtctt?caggtttcaa?aatcaacagt????120
gcaaaagatg?acgcagccgg?tctgcaaatc?tctaaccgct?tgaacgtaca?aagtcgcggt????180
ctagacgttg?cggtacgtaa?cgccaacgca?ggtatctcaa?tcgcacaaac?cgcagaaggt????240
gcgatgaacg?agaccaccaa?catcctacaa?cgtatgcgtg?acctatctct?acaatccgcg????300
aacggctcaa?actcaaaatc?agagcgcgtg?gcgattcaag?aagaagtgac?agcattgaat????360
gacgagctaa?accgtattgc?agaaaccacg?tcttttggtg?gtgacaagct?gctaaacggt????420
acttacggca?cgaaagcaat?gcaaattggt?gcggataacg?gtgaagcggt?catgctttca????480
ctgaaagaca?tgcgctctga?caacgtgatg?atgggcggcg?tgagctacca?agctgaagaa????540
ggcaaagaca?agaactggaa?tgtggccgca?ggcgacaacg?acttgacgat?tgcactgaca????600
gacagctttg?gtaacgagca?agagatcgaa?atcaacgcga?aagcgggtga?tgacatcgaa????660
gagctagcga?cgtacatcaa?cggtcaaact?gaccttgtaa?aagcgtcagt?gggtgaaggc????720
ggcaagctac?agatctttgc?tggtaacaac?aaagttcaag?gtgaaattgc?tttctcaggt????780
agcctagctg?gtgaacttgg?cctaggcgaa?ggcaaaaacg?tcacggtaga?cacgattgac????840
gtgacaaccg?tacaaggtgc?gcaagagtcg?gtagcgattg?tggatgcggc?actgaaatac????900
gtagacagcc?accgtgcaga?gctgggtgca?ttccagaacc?gtttcaacca?tgcaatcagc????960
aacttggaca?acatcaacga?aaacgtgaac?gcgtcgaaga?gccgaatcaa?agataccgac????1020
ttcgcgaaag?aaacgactca?gttgaccaag?acacaaattc?tatcgcaagc?atcaagttcc????1080
attcttgcgc?aagcgaaaca?agcgccaaac?tcagcgctaa?gtctactagg?ctaa??????????1134
<210>44
<211>377
<212>PRT
＜213〉Vibrio vulnificus D70A/N135D FlaB amino acid
<400>44
Met?Ala?Val?Asn?Val?Asn?Thr?Asn?Val?Ala?Ala?Met?Thr?Ala?Gln?Arg
1???????????????5??????????????????10??????????????????15
Tyr?Leu?Asn?Asn?Ala?Asn?Ser?Ala?Gln?Gln?Thr?Ser?Met?Glu?Arg?Leu
20??????????????????25??????????????????30
Ser?Ser?Gly?Phe?Lys?Ile?Asn?Ser?Ala?Lys?Asp?Asp?Ala?Ala?Gly?Leu
35??????????????????40??????????????????45
Gln?Ile?Ser?Asn?Arg?Leu?Asn?Val?Gln?Ser?Arg?Gly?Leu?Asp?Val?Ala
50??????????????????55??????????????????60
Val?Arg?Asn?Ala?Asn?Ala?Gly?Ile?Ser?Ile?Ala?Gln?Thr?Ala?Glu?Gly
65??????????????????70??????????????????75??????????????????80
Ala?Met?Asn?Glu?Thr?Thr?Asn?Ile?Leu?Gln?Arg?Met?Arg?Asp?Leu?Ser
85??????????????????90??????????????????95
Leu?Gln?Ser?Ala?Asn?Gly?Ser?Asn?Ser?Lys?Ser?Glu?Arg?Val?Ala?Ile
100?????????????????105?????????????????110
Gln?Glu?Glu?Val?Thr?Ala?Leu?Asn?Asp?Glu?Leu?Asn?Arg?Ile?Ala?Glu
115?????????????????120?????????????????125
Thr?Thr?Ser?Phe?Gly?Gly?Asp?Lys?Leu?Leu?Asn?Gly?Thr?Tyr?Gly?Thr
130?????????????????135?????????????????140
Lys?Ala?Met?Gln?Ile?Gly?Ala?Asp?Asn?Gly?Glu?Ala?Val?Met?Leu?Ser
145?????????????????150?????????????????155?????????????????160
Leu?Lys?Asp?Met?Arg?Ser?Asp?Asn?Val?Met?Met?Gly?Gly?Val?Ser?Tyr
165?????????????????170?????????????????175
Gln?Ala?Glu?Glu?Gly?Lys?Asp?Lys?Asn?Trp?Asn?Val?Ala?Ala?Gly?Asp
180?????????????????185?????????????????190
Asn?Asp?Leu?Thr?Ile?Ala?Leu?Thr?Asp?Ser?Phe?Gly?Asn?Glu?Gln?Glu
195?????????????????200?????????????????205
Ile?Glu?Ile?Asn?Ala?Lys?Ala?Gly?Asp?Asp?Ile?Glu?Glu?Leu?Ala?Thr
210?????????????????215?????????????????220
Tyr?Ile?Asn?Gly?Gln?Thr?Asp?Leu?Val?Lys?Ala?Ser?Val?Gly?Glu?Gly
225?????????????????230?????????????????235?????????????????240
Gly?Lys?Leu?Gln?Ile?Phe?Ala?Gly?Asn?Asn?Lys?Val?Gln?Gly?Glu?Ile
245?????????????????250?????????????????255
Ala?Phe?Ser?Gly?Ser?Leu?Ala?Gly?Glu?Leu?Gly?Leu?Gly?Glu?Gly?Lys
260?????????????????265?????????????????270
Asn?Val?Thr?Val?Asp?Thr?Ile?Asp?Val?Thr?Thr?Val?Gln?Gly?Ala?Gln
275?????????????????280?????????????????285
Glu?Ser?Val?Ala?Ile?Val?Asp?Ala?Ala?Leu?Lys?Tyr?Val?Asp?Ser?His
290?????????????????295?????????????????300
Arg?Ala?Glu?Leu?Gly?Ala?Phe?Gln?Asn?Arg?Phe?Asn?His?Ala?Ile?Ser
305?????????????????310?????????????????315?????????????????320
Asn?Leu?Asp?Asn?Ile?Asn?Glu?Asn?Val?Asn?Ala?Ser?Lys?Ser?Arg?Ile
325?????????????????330?????????????????335
Lys?Asp?Thr?Asp?Phe?Ala?Lys?Glu?Thr?Thr?Gln?Leu?Thr?Lys?Thr?Gln
340?????????????????345?????????????????350
Ile?Leu?Ser?Gln?Ala?Ser?Ser?Ser?Ile?Leu?Ala?Gln?Ala?Lys?Gln?Ala
355?????????????????360?????????????????365
Pro?Asn?Ser?Ala?Leu?Ser?Leu?Leu?Gly
370?????????????????375

Claims (14)

1, a kind of Vibrio vulnificus flagellin mutant, this flagellin mutant has enhanced toll sample acceptor (TLR-5) stimulating activity.
2, Vibrio vulnificus flagellin mutant according to claim 1, wherein, this flagellin mutant is the flagellin mutant (D70A) shown in the SEQ ID NO:2, and this flagellin mutant (D70A) is by becoming L-Ala (A) to prepare aspartic acid (D70) site-directed mutagenesis on the 70th of the Vibrio vulnificus flagellin FlaB.
3, Vibrio vulnificus flagellin mutant according to claim 1, wherein, this flagellin mutant is the flagellin mutant (R93A) shown in the SEQ ID NO:4, and this flagellin mutant (R93A) is by becoming L-Ala (A) to prepare arginine (R93) site-directed mutagenesis on the 93rd of the Vibrio vulnificus flagellin FlaB.
4, Vibrio vulnificus flagellin mutant according to claim 1, wherein, this flagellin mutant is the flagellin mutant (L97W) shown in the SEQ ID NO:6, and this flagellin mutant (L97W) is by becoming tryptophane (W) to prepare leucine (L97) site-directed mutagenesis on the 97th of the Vibrio vulnificus flagellin FlaB.
5, Vibrio vulnificus flagellin mutant according to claim 1, wherein, this flagellin mutant is the flagellin mutant (N101A) shown in the SEQ ID NO:8, and this flagellin mutant (N101A) is by becoming L-Ala (A) to prepare l-asparagine (N101) site-directed mutagenesis on the 101st of the Vibrio vulnificus flagellin FlaB.
6, Vibrio vulnificus flagellin mutant according to claim 1, wherein, this flagellin mutant is the flagellin mutant (S103W) shown in the SEQ ID NO:10, and this flagellin mutant (S103W) is by becoming tryptophane (W) to prepare Serine (S103) site-directed mutagenesis on the 103rd of the Vibrio vulnificus flagellin FlaB.
7, Vibrio vulnificus flagellin mutant according to claim 1, wherein, this flagellin mutant is the flagellin mutant (E108A) shown in the SEQ ID NO:12, and this flagellin mutant (E108A) is by becoming L-Ala (A) to prepare L-glutamic acid (E108) site-directed mutagenesis on the 108th of the Vibrio vulnificus flagellin FlaB.
8, Vibrio vulnificus flagellin mutant according to claim 1, wherein, this flagellin mutant is the flagellin mutant (N135D) shown in the SEQ ID NO:14, and this flagellin mutant (N135D) is by becoming aspartic acid (D) to prepare l-asparagine (N135) site-directed mutagenesis on the 135th of the Vibrio vulnificus flagellin FlaB.
9, Vibrio vulnificus flagellin mutant according to claim 1, wherein, this flagellin mutant is the flagellin mutant (A151W) shown in the SEQ ID NO:16, and this flagellin mutant (A151W) is by becoming tryptophane (W) to prepare L-Ala (A151) site-directed mutagenesis on the 151st of the Vibrio vulnificus flagellin FlaB.
10, Vibrio vulnificus flagellin mutant according to claim 1, wherein, this flagellin mutant is the flagellin mutant (N153W) shown in the SEQ ID NO:18, and this flagellin mutant (N153W) is by becoming tryptophane (W) to prepare l-asparagine (N153) site-directed mutagenesis on the 153rd of the Vibrio vulnificus flagellin FlaB.
11, Vibrio vulnificus flagellin mutant according to claim 1, wherein, this flagellin mutant is the flagellin mutant (V291W) shown in the SEQ ID NO:20, and this flagellin mutant (V291W) is by becoming tryptophane (W) to prepare Xie Ansuan (V291) site-directed mutagenesis on the 291st of the Vibrio vulnificus flagellin FlaB.
12, Vibrio vulnificus flagellin mutant according to claim 1, wherein, this flagellin mutant is the flagellin mutant (D70A/N135D) shown in the SEQ ID NO:44, and this flagellin mutant (D70A/N135D) is by aspartic acid (D70) site-directed mutagenesis on the 70th of the Vibrio vulnificus flagellin FlaB being become L-Ala (A) and becoming aspartic acid (D) to prepare l-asparagine (N135) site-directed mutagenesis on the 135th of the Vibrio vulnificus flagellin FlaB.
13, a kind of vaccine adjuvant is characterized in that, this vaccine adjuvant contains at least a being selected from by the flagellin mutant in SEQID NO:2,4,6,8,10,12,14,16,18,20 and 44 groups of being formed as activeconstituents.
14, the flagellin mutant is used to stimulate the active purposes of toll sample receptor-5, wherein, described flagellin mutant is at least a being selected from by the flagellin mutant in SEQ ID NO:2,4,6,8,10,12,14,16,18,20 and 44 groups of being formed.
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KR1020080011330A KR100960611B1 (en) 2008-02-04 2008-02-04 Modified flagellin improved Toll-like receptor 5 stimulating activity
KR10-2008-0011330 2008-02-04
KR1020080011330 2008-02-04
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106362144A (en) * 2016-10-31 2017-02-01 中国科学院武汉病毒研究所 Respiratory syncytial virus (RSV) vaccine
CN108997491A (en) * 2018-06-26 2018-12-14 中山大学 Epinephelus coioides innate immunity receptor TLR5S gene and its new application for encoding albumen

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US6211159B1 (en) 1997-04-11 2001-04-03 University Of Toronto Flagellin gene, FlaC of campylobacter
KR20050073865A (en) * 2004-01-12 2005-07-18 대한민국(전남대학교총장) Vaccine adjuvants containing constituents of the flagellum of vibrio vulnificus as an active component
MY157941A (en) * 2005-01-19 2016-08-15 Vaxinnate Corp Compositions of pathogen-associated molecular patterns and methods of use
KR100804507B1 (en) * 2005-05-17 2008-02-20 전남대학교산학협력단 Adjuvant compositions comprising flagellins useful for immunotherapy of asthma and allergic diseases

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106362144A (en) * 2016-10-31 2017-02-01 中国科学院武汉病毒研究所 Respiratory syncytial virus (RSV) vaccine
CN108997491A (en) * 2018-06-26 2018-12-14 中山大学 Epinephelus coioides innate immunity receptor TLR5S gene and its new application for encoding albumen

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