CN101622272B - Modified flagellin improved toll-like receptor 5 stimulating activity - Google Patents
Modified flagellin improved toll-like receptor 5 stimulating activity Download PDFInfo
- Publication number
- CN101622272B CN101622272B CN2008800040410A CN200880004041A CN101622272B CN 101622272 B CN101622272 B CN 101622272B CN 2008800040410 A CN2008800040410 A CN 2008800040410A CN 200880004041 A CN200880004041 A CN 200880004041A CN 101622272 B CN101622272 B CN 101622272B
- Authority
- CN
- China
- Prior art keywords
- flagellin
- seq
- mutant
- flab
- directed mutagenesis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (F)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/107—Vibrio
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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
Technical field
The present invention relates to have the flagellin mutant of toll sample receptor-5 (hereinafter referred to as " the TLR5 ") stimulating activity of enhancing, particularly the flagellin mutant that is prepared 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 that determines 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 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 identify 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, Aderem A: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 the vaccine co-administered time, can strengthen, prolongs or accelerate the material of the Ag-specific immune response of being induced by vaccine antigen.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.
Desirable vaccine adjuvant: must be safe 1); 2) must be biodegradable in vivo; Compare when 3) individually dosed with antigen, must show that effectively preventative or therapeutic immunization is replied; 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 the material of vaccine adjuvant as vaccine adjuvant at present: 1) mineral salt, for example aluminum hydroxide gel; 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 the heat-labile toxin (LT) that Toxins,exo-, cholera (CT) that vibrio cholerae (Vibrio cholerae) derives and intestinal bacteria (Escherichia coli) derive 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 the 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 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 with TLR5 stimulating activity of enhancing by the reorganization flagellin of Sustainable Control.
According to the 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.20034 (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 have been found that the flagellin mutant of preparation compares the TLR5 stimulating activity with remarkable enhancing with existing (wild-type) flagellin, thereby finished the present invention.
Therefore, the flagellin mutant that the purpose of this invention is to provide the TLR5 stimulating activity with enhancing.
Another object of the present invention provides and contains at least a described flagellin mutant as the vaccine adjuvant of activeconstituents.
For achieving the above object, the invention provides for being suppressed at interactional flagellin mutant between the TLR5 agonist flagellin flagellin monomer.
The present invention also provides and contains at least a described flagellin mutant as the vaccine adjuvant of activeconstituents.
In the present invention, by being provided, the flagellin mutant of comparing the TLR5 stimulating activity with enhancing with existing flagellin developed the flagellin vaccine adjuvant of improvement, 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 etc., 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.2001410 (6826): 331-7).
Fig. 2 is presented to make up in the flagellin mutant process of the present invention, induce synoptic diagram (the Samatey FA of the strategy of rite-directed mutagenesis at Vibrio vulnificus flagellin gene flaB, Deng the people, Structure ofthe bacterial flagellar protofilament and implications for a switch for supercoiling.Nature.2001410 (6826): 331-7).
Fig. 3 and 4 shows that reorganization flagellin mutant of the present invention is to the influence of the flagellin polymerization 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 poly cooperation usefulness of flagellin monomer, thereby this flagellin mutant shows the TLR5 stimulating activity of enhancing.
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 flagellin FlaB has participated in the axial interaction between the flagellin monomer in the TLR5 agonist flagellin according to one's analysis; 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, has the flagellin mutant of 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 the TLR5 stimulating activity of remarkable enhancing, 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 TLR5 stimulating activity of remarkable enhancing prepared in accordance with the present invention can provide more effective TLR agonist replacing existing flagellin, thereby the vaccine adjuvant of comparing the effectiveness with enhancing with existing flagellin is provided.
Prove that the method that flagellin mutant of the present invention has a TLR5 stimulating activity of enhancing 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 the TLR5 activation of the flagellin mutant of respectively recombinating in intestinal bacteria, induce.
Embodiment
Hereinafter with reference to embodiment and test implementation example the present invention is described in more detail.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::T7genel 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.2003 424 (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.2001 410 (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.Namely be respectively the L-Ala (A151) of the 153rd of the aspartic acid (D70) of the 70th of the last subunit (upper subunit) that has participated in axial interactional each monomer, 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 the following subunit (lower subunit) of each monomer, 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 namely 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 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 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 the dna profiling for 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 for the 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 for the 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.
Being used for the PCR condition of structure Vibrio vulnificus flagellin gene flaB directed mutants and clone's plasmid sums 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 |
| A15lW | 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 |
| V29lW | 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 by supplier's explanation and come synthetic sudden change chain.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 the 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 the point mutation albumen that separates.Use Superdex 120 posts (AKTA-Prime Amersham) carries out gel-filtration to the recombinant protein that separates, 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 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, 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, by 1 * 10
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 SEQ ID NO:2,4,6,8,10,12,14,16,18,20 and 44 of embodiment 1 separation.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 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.
Claims (2)
1. Vibrio vulnificus flagellin mutant, this flagellin mutant has the toll sample receptor-5 stimulating activity of enhancing; This flagellin mutant is the flagellin mutant shown in the SEQ ID NO:2,4,6,8,10,12,14,16,18,20 or 44.
2. a vaccine adjuvant is characterized in that, this vaccine adjuvant contains 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 forming as activeconstituents.
Applications Claiming Priority (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2007-0013846 | 2007-02-09 | ||
| KR1020070013846 | 2007-02-09 | ||
| KR1020070013846A KR100823866B1 (en) | 2007-02-09 | 2007-02-09 | - -5 Modified flagellin improved Toll-like receptor 5 stimulating activity |
| 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 | ||
| PCT/KR2008/000709 WO2008097016A1 (en) | 2007-02-09 | 2008-02-05 | Modified flagellin improved toll-like receptor 5 stimulating activity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101622272A CN101622272A (en) | 2010-01-06 |
| CN101622272B true CN101622272B (en) | 2013-07-17 |
Family
ID=39572082
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008800040410A Active CN101622272B (en) | 2007-02-09 | 2008-02-05 | Modified flagellin improved toll-like receptor 5 stimulating activity |
Country Status (2)
| Country | Link |
|---|---|
| KR (1) | KR100823866B1 (en) |
| CN (1) | CN101622272B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106362144B (en) * | 2016-10-31 | 2021-02-09 | 武汉三利生物技术有限公司 | Respiratory syncytial virus 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005070455A1 (en) * | 2004-01-12 | 2005-08-04 | Chonnam National University | Mucosal vaccine adjuvants containing bacterial flegellins as an active component |
| WO2006078657A2 (en) * | 2005-01-19 | 2006-07-27 | Vaxinnate Corporation | Compositions comprising pathogen-associated molecular patterns and antigens and their use to stimulate an immune response |
| KR20060118737A (en) * | 2005-05-17 | 2006-11-24 | 프로셀주식회사 | Adjuvant compositions comprising flagellins useful for immunotherapy of asthma and allergic diseases |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6211159B1 (en) | 1997-04-11 | 2001-04-03 | University Of Toronto | Flagellin gene, FlaC of campylobacter |
-
2007
- 2007-02-09 KR KR1020070013846A patent/KR100823866B1/en active IP Right Grant
-
2008
- 2008-02-05 CN CN2008800040410A patent/CN101622272B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005070455A1 (en) * | 2004-01-12 | 2005-08-04 | Chonnam National University | Mucosal vaccine adjuvants containing bacterial flegellins as an active component |
| WO2006078657A2 (en) * | 2005-01-19 | 2006-07-27 | Vaxinnate Corporation | Compositions comprising pathogen-associated molecular patterns and antigens and their use to stimulate an immune response |
| KR20060118737A (en) * | 2005-05-17 | 2006-11-24 | 프로셀주식회사 | Adjuvant compositions comprising flagellins useful for immunotherapy of asthma and allergic diseases |
Also Published As
| Publication number | Publication date |
|---|---|
| KR100823866B1 (en) | 2008-04-21 |
| CN101622272A (en) | 2010-01-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2121734B1 (en) | Modified flagellin improved toll-like receptor 5 stimulating activity | |
| IL151942A (en) | Method for enhancing immunogenicity of tumor antigens | |
| CA2413045A1 (en) | Expression system | |
| US20090297556A1 (en) | Salmonella Based Oral Vaccines for Anthrax | |
| CN101622272B (en) | Modified flagellin improved toll-like receptor 5 stimulating activity | |
| EP3299384B1 (en) | Truncated rotavirus vp4 protein and application thereof | |
| WO2020220846A1 (en) | Fusion protein capable of self-assembling into protein nanoparticles and application thereof | |
| KR101130884B1 (en) | Recombinant fusion protein produced by fusing Vibrio vulnificus flagellin and pathogenic antigens and the mucosal vaccine containing the same as an active ingredient | |
| CN108578686A (en) | A method of preparing clostridiosis of sheep genetic engineering subunit vaccine | |
| KR100854594B1 (en) | - -5 - PAS-flagellin fusion protein with improved Toll-like receptor 5 stimulating activity | |
| Angelos et al. | Systemic and ocular immune responses in cattle following intranasal vaccination with precipitated or partially solubilized recombinant Moraxella bovis cytotoxin adjuvanted with polyacrylic acid | |
| KR100960611B1 (en) | Modified flagellin improved Toll-like receptor 5 stimulating activity | |
| CN110922454A (en) | Immune application of pseudomonas aeruginosa toxin ExoS and ExoT and preparation method thereof | |
| CN110141661A (en) | Botulinal toxin A Hc vaccine liquid aersol lung delivers immune mouse model | |
| AU778957B2 (en) | Bacterial membrane fractions with adjuvant effect | |
| JP6039648B2 (en) | Combination vaccine | |
| KR102259933B1 (en) | Method for manufacturing anthrax spore vaccines using spore surface display and anthrax spore vaccines | |
| WO2021120019A1 (en) | Composition for immunization and preparation method therefor | |
| KR102105021B1 (en) | Vaccine composition for preventing or treating porcine edema disease comprising Stx2eA-C-terminal fragment-(Stx2eB)5 recombinant protein as an antigen | |
| Boyd | Design and Production of a Recombinant FliC-Antigen Co-Expression Platform for Increased Vaccine Efficacy | |
| CN116478303A (en) | Fusion protein capable of self-assembling into nano particles and application thereof in immunization | |
| Moigne et al. | Homologous recombination with linear DNA to insert antigenic protein in the flagellin: improvement of the Th1 immune response | |
| CN111729080A (en) | Biological particle type composite adjuvant and preparation method and application thereof | |
| CN111150839A (en) | Composition for immunization and preparation method thereof | |
| CN115838431A (en) | Fusion protein KP-Ag2 used as Klebsiella pneumoniae vaccine antigen and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210204 Address after: Kwangju, South Korea Patentee after: Li Junxing Patentee after: Li Shien Address before: Kwangju, South Korea Patentee before: INDUSTRY FOUNDATION OF CHONNAM NATIONAL University |