CN117866060A - Bacterial flagellin lacking hypervariable region structural domain, and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a bacterial flagellin with a deleted hypervariable region structural domain, a preparation method and application thereof, wherein the bacterial flagellin with the deleted hypervariable region structural domain is compared with fliC with an intact structural domain H7 The 178 th amino acid to 497 th amino acid of the hypervariable region is deleted. The invention constructs truncated recombinant E.coli flagellin fliC by deleting the intermediate hypervariable region of the flagellin NC Detection by in vitro Caco-2 cell modelDiscovery of FliC NC Has a function of FliC H7 Similar and good TLR5 receptor activity, can activate the TLR5 signaling pathway; in vivo, fliC NC Recombinant flagellin is also capable of inducing high levels of IgG antibodies against the patterned antigen FaeG in vivo and humoral and cellular immunity-related inflammatory genes in spleen cells.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a bacterial flagellin lacking a hypervariable region structural domain, and a preparation method and application thereof.
Background
Vaccine immunization is one of the most economical, effective and safe means for preventing and controlling various infectious diseases, and vaccination can effectively reduce the morbidity and mortality of diseases. By vaccination, the body can obtain immune protection against specific pathogens, greatly reducing the risk of infection. However, inactivated vaccine, protein subunit vaccine, nucleic acid vaccine and the like have weak immunogenicity of the antigen itself, and an immune adjuvant is required to be added to enhance the immunogenicity and improve the immune protection effect. Some traditional immunoadjuvants such as aluminum hydroxide gel, surfactant, white oil adjuvant and the like can enhance the immunogenicity of antigens to different degrees, but still have many limitations such as limited immune pathways, insignificant immune effects, mainly induction of humoral immune response, relatively high cost, low safety and the like. Therefore, a novel immune adjuvant which is safe, has excellent immune adjuvant effect and has low cost needs to be developed.
In recent years, many pathogenic microorganisms or components thereof have been found to have excellent immunoadjuvant effects, and among them, toll-like receptor (TLRs) ligands have been attracting attention as adjuvant development. Bacterial flagellin is a ligand of TLR5, is used as a novel immunoadjuvant, and has strong immunoadjuvant activity. It not only can activate the inherent immune response system of the organism, but also can induce cellular immunity and humoral immunity at the same time. Since McEwen first demonstrated the immunoadjuvant effect of flagellin, the structural basis, molecular mechanism, etc. of the flagellin to exert the immunoadjuvant effect have been one of the leading edge and hot spot problems of research in the International scientific community.
Analysis of the crystal structure of salmonella flagellin using electron microscopy and X-ray diffraction showed that the intact flagellin comprises four related domains D0, D1, D2 and D3. Different domains of flagellin have different structural features and perform different biological functions. The D0 and D1 domains located at the NH2 and COOH ends of flagellin are highly conserved among different species, and can activate TLR5 and/or NAIP5-NLRC4 signaling pathways, exerting an immunoadjuvant effect. The intermediate hypervariable region (V) comprises D2 and D3 domains, the D3 domains having a high degree of variability due to exposure to the surface of the flagella filaments. The hypervariable regions of flagellin mainly determine the serotypes and autoimmune properties of the flagella, and are also essential for flagellin antigenicity. However, the presence of hypervariable regions not only induces the body to produce specific antibodies against itself, but also induces antibodies against flagellin that may neutralize TLR5 receptor signaling and reduce the immune effects of flagellin. In addition, the presence of hypervariable regions can also induce host cytotoxicity and other problems. The structural characteristics of different domains of the flagellin are further clarified by utilizing structural biology, and important information can be provided for deeply researching functions of the flagellin.
Bacterial flagellin can activate the body's innate immune system through TLR5 and NLRC4 signaling pathways, induce the secretion of pro-inflammatory factors (IL-1 beta, IL-8, IL-18, TNF-alpha, etc.), and thereby exert an immunoadjuvant effect. Extracellular flagellin can be bound to and activate Toll-like receptor 5 (TLR 5) on the cell surface, and induce secretion of inflammatory factors such as IL-8, TNF-alpha and the like; the intracellular flagellin then binds to and activates the NAIP5-NLRC4 complex, inducing IL-1β, IL-18 production and apoptosis. Cytokines and chemokines induced by flagellin through TLR5 and/or NLRC4 signaling pathways are key to the activation of innate and acquired immunity by flagellin to exert immune adjuvants during infection by pathogenic microorganisms.
Flagellin has a number of advantages as a novel molecular immunoadjuvant: 1) Flagellin not only induces humoral immunity, but also induces high levels of cellular immune response. 2) Flagellin can bind to foreign antigens in a variety of forms (mixed/chimeric/fused) and exert an adjuvant effect through a variety of immune pathways. 3) The protein structure has strong plasticity, and the insertion of exogenous antigen at NH2 end, COOH terminal or hypervariable region does not affect the structure and TLR5 receptor activity. 4) Flagellin can overcome oral tolerance and efficiently induce pro-inflammatory cytokines on mucosal surfaces. 5) In addition to acting as an immunoadjuvant itself, flagellin also acts as an enhancer of other immunoadjuvants. Although bacterial flagellin has many advantages as a novel adjuvant, there is currently no commercially available or approved vaccine based on flagellin as an adjuvant, and only recombinant fused influenza-flagellin vaccines have entered phase II clinical studies. The main reason for this is that the body-induced antibodies against the flagellin itself may interfere with the re-immune response due to the use of intact bacterial flagellin as an adjuvant, and the safety of the intact flagellin as an adjuvant is yet to be further evaluated. Therefore, development, improvement and optimization of immune adjuvant-based flagellin in combination with structural biology of bacterial flagellin is imperative.
Disclosure of Invention
The invention aims to: the invention aims to provide an escherichia coli flagellin (FliC) which has excellent adjuvant effect, is safe and efficient and lacks an intermediate hypervariable region NC ) And a preparation method and application thereof, and overcomes the problems faced by the flagellin used as an immunoadjuvant in clinical application. By truncating the intermediate hypervariable region (V), only the conserved NH 2-and COOH-termini thereof associated with the immunoadjuvant effect are retained. Flagellin mutant (FliC) deleted of hypervariable region NC ) The anti-tumor antibody has the TLR5 receptor activity of full-length flagellin, and avoids the inherent antigenicity of the flagellin, thereby preventing the secondary immune response or TLR5 signal crosstalk of the organism from being influenced by the induction of specific antibodies aiming at the flagellin by the presence of a hypervariable region. In addition, recombinant flagellin FliC NC Does not cause any significant systemic pro-inflammatory response after the immune injection into the body, thereby having the potential to be developed into a safer, efficient and side-effect free bacterial flagellin immunoadjuvant.
The technical scheme is as follows: in order to solve the technical problems, the invention provides a bacterial flagellin with a deleted hypervariable region domain, which is compared with FliC with an intact domain H7 The 178 th amino acid to 497 th amino acid of the hypervariable region is deleted.
Wherein the amino acid sequence of the mutant protein is shown as SEQ ID NO. 2.
The invention also comprises a mutant gene of the bacterial flagellin for encoding the deletion hypervariable region structural domain, wherein the mutant gene is the deletion of 534 to 1491 bases of the hypervariable region of the fliC full-length gene of the escherichia coli H7 serotype.
Wherein, the nucleotide sequence of the mutant gene of the bacterial flagellin deleted with the hypervariable region structural domain is shown as SEQ ID NO. 1.
The invention also includes an expression cassette, a recombinant vector, a recombinant cell or a recombinant bacterium, which contains the mutant gene.
The invention also discloses a preparation method of the bacterial flagellin with the deleted hypervariable region structural domain, which comprises the following steps: deletion of the bases 534-1491 of the high-variable region of fliC full-length gene for encoding the H7 serotype of the escherichia coli, cloning to a prokaryotic expression vector to construct a recombinant expression plasmid, and transforming the recombinant expression plasmid into the escherichia coli engineering bacteria to perform prokaryotic efficient expression of fliC after IPTG induction NC Recombinant protein is obtained.
FliC of the invention NC The recombinant protein lacks a hypervariable region related to flagellin antigenicity, and can not be subjected to rabbit anti-FliC in Western blot experiments H7 Polyclonal antibody recognition, indicating deletion of the hypervariable region FliC NC The recombinant human flagellin has no antigenicity of flagellin, and can effectively avoid interference of existence of flagellin autoantibodies on re-immune response of organisms. FliC NC Recombinant proteins are compared to FliC with intact domains H7 FliC retaining only NH 2-and COOH-ends NC Has a corresponding TLR5 receptor stimulating activity in vitro. FliC NC Has good adjuvant activity in vivo. In FliC NC As an immunoadjuvant with a model antigen FaThe eG mixed immunization of BALB/c mice was able to induce high titres of anti-FaeG IgG antibodies.
Wherein the GeneBank accession number of the fliC full-length gene encoding the E.coli H7 serotype is AY249992.
The invention also comprises the bacterial flagellin deleted of the hypervariable region structural domain, the mutant gene and the application of the expression cassette, the recombinant vector, the recombinant cell or the recombinant bacterium in preparing vaccine immunoadjuvant.
The invention also includes a vaccine immunoadjuvant comprising the bacterial flagellin lacking the hypervariable region domain or the mutant gene.
Wherein the vaccine immunoadjuvant further comprises a model antigen FaeG antigen.
FliC of the invention NC The recombinant protein and the model antigen FaeG antigen are mixed to immunize BALB/c mice, so that spleen Th1 (TNF-alpha and IFN-gamma) immune response and Th2 (IL-4) immune response can be induced, and Th1 (cell) immune response is the main.
In view of this deletion of the intermediate hypervariable region, only the FliC of the conserved domain is retained NC The flagella recombinant protein has excellent immunological adjuvant effect and FliC NC The recombinant flagellin can be used as an immunoadjuvant for the research and development of vaccines against various pathogens such as bacteria, viruses and parasites.
The preparation and application of the recombinant flagellin deleted of the intermediate hypervariable region are also applicable to other serotypes of flagellin of escherichia coli and other bacterial flagellins.
The invention uses GPVD linker to connect the conserved NH 2-end and COOH end at two ends after shortening the intermediate hypervariable region (V) of the flagellin of the Escherichia coli H7 serotype, and uses SOE-PCR technology to amplify the FliC NC Construction of truncated recombinant flagellin FliC NC 。
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: fliC constructed according to the invention NC Recombinant flagellin has a similar spatial conformation and biological activity as native flagellin; not only can induce humoral immune response against target antigen, but also can induce cellular immunityReacting; the immunity effect is effectively improved, and the bottleneck of the existing bacterial flagellin-based immune adjuvant is innovatively improved and overcome. The flagellin conserved domain is expected to be used as a novel molecular adjuvant to be applied to the research and development of vaccines for various diseases such as clinical bacteria, viruses, parasites and the like.
Drawings
FIG. 1 is fliC NC Schematic construction of recombinant bacterial flagellin. Wherein N represents the NH2 terminus of flagellin; v represents the hypervariable region in the middle of flagellin; c represents the COOH end of flagellin, and the specific amino acid sequence of Linker is GPGPG.
FIG. 2 shows fliC full length and fliC NC PCR amplification identification electropherograms of genes. Wherein lane M is a 2K Plus II DNA Marker; lane 1 is fliC H7 A full-length gene; lane 2 is fliC NC And (3) a gene.
FIG. 3 is a recombinant fliC NC SDS-PAGE and Western blot identification of flagellin. A. Recombinant FliC NC SDS-PAGE electrophoresis of flagellin. Wherein lane M is a Protein marker; lane 1 is fliC H7 Recombinant flagellin; lane 2 is fliC NC Recombinant flagellin. B. Recombinant FliC NC Western blot identification of flagellin. Lane 1 is fliC H7 Recombinant flagellin; lane 2 is fliC NC Recombinant flagellin.
FIG. 4 is a fliC NC And detecting the activity of the TLR5 receptor in vitro of the recombinant flagellin. FliC from which endotoxin had been removed with 5 μg/mL H7 And FliC NC The recombinant flagellin stimulates human colon cancer cells Caco-2 for 6h respectively, wherein unstimulated Caco-2 cells are used as a negative control group. Cell culture supernatants were collected and the concentrations of IL-8 and TNF- α in the cell supernatants were measured using human IL-8 and TNF- α ELISA kits, respectively.
FIG. 5 is a graph showing the detection of the antibody level of the model antigen FaeG in serum of immunized mice by ELISA. FliC is respectively carried out H7 、FliC NC Subcutaneously immunizing mice with recombinant flagellin and Freund's adjuvant as immunoadjuvant and model antigen FaeG (enterotoxigenic Escherichia coli F4ac pilus adhesion main subunit), and detecting each with the extracted F4ac pilus adhesion as coating antigenIgG antibody titer against FaeG in serum of group immunized mice, with the immunized PBS group as the negative control group.
FIG. 6 is a diagram showing the RT-PCR detection of inflammatory cytokine mRNA expression in spleen cells of immunized mice. RT-PCR was performed to detect the expression levels of the il-4, tnf-alpha and inf-gamma genes in spleen cells of each group of immunized mice, respectively. A. Expression of the il-4 gene in spleen cells. B. Expression of the tnf-alpha gene in spleen cells. C. Expression of the inf-gamma gene in spleen cells.
Detailed Description
Embodiments of the present invention will now be described more fully hereinafter with reference to the accompanying examples, in which it is shown, however, to illustrate some of the examples of the invention, and not to limit the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were not manufacturer-specific and were considered conventional products available commercially.
Example 1
1. Coli flagellin fliCNC gene clone and recombinant plasmid construction
(1)FliC NC Construction and structure prediction of (c)
Modeling, prediction and analysis of FliC using Phyre2 portal NC Secondary structure of the amino acid sequence of the flagellin mutant. FliC protein (UniProt accession number: A0A2Z5FPT 6) was used as a control, and the results showed that FliC NC The protein remains in a native and relatively independent spatial conformation.
(2) Primer design and Gene amplification
Amplification primers, fliC, were designed using DNASTRA software based on the gene sequence encoding the flagellin fliC of E.coli with hemorrhagic properties as published in GenBank (GenBank accession number: AY 249992) H7 -F (SEQ ID NO. 3) and fliC H7 -R (SEQ ID NO. 4) for PCR amplification of fliC H7 Full length of the gene; primer FliC H7 -F (SEQ ID NO. 3) and fliC NC -R (SEQ ID NO. 5) for PCR amplification of fliC NC The gene and reaction amplification system are shown in Table 1, and the PCR reaction conditions are: pre-denaturation at 95℃for 5min, denaturation at 94℃for 30s, annealing at 55℃for 30s, and extension at 72 ℃2min,30 cycles, 72 ℃ x 10min, and 4 ℃. The upstream and downstream primers have BamHI-HF and SalI-HF cleavage sites, respectively. Extracting genome from enterohemorrhagic Escherichia coli EHEC (O157: H7) by boiling lysis method, and PCR amplifying fliC with Phusion High-Fidelity DNA polymerase (Beijing full-size gold organism, cat# AP 221-12) H7 And fliC NC A gene fragment. The results are shown in FIG. 2, fliC H7 Full-length gene and fliC NC The gene fragment sizes are about 1785bp and 807bp respectively, which are in accordance with the expected result.
TABLE 1 PCR amplification System
(3) Construction of pET-28 alpha (+) -fliCNC recombinant plasmid
fliC is to be fliC H7 And fliC NC The PCR recovery of the purified gene fragment and the plasmid pET-28. Alpha. (+) vector were double digested with BamHI-HF (NEB Co., ltd., cat# R3136) and SalI-HF (NEB Co., ltd., cat# R3138) in a 37℃water bath for 2 hours, respectively, and the double digested reaction system was as shown in Table 2. The enzyme-cut product is purified and recovered, then is connected at 16 ℃ for overnight by using T4 DNA ligase, the connection product is transformed into DH5 alpha competent cells, then the DH5 alpha competent cells are added into LB liquid medium for shaking culture for 1h, a proper amount of culture product is coated on LB solid medium of kanamycin (Kan+) and cultured overnight in a bacterial constant temperature incubator at 37 ℃, a plurality of single colonies are picked up the next day, are correctly identified by PCR and then are sent to a company for sequencing, finally, the correct cloning construction is confirmed by DNA sequencing, and the recombinant plasmid is named as pET-28 alpha (+) -fliC NC 。
TABLE 2 cleavage reaction System
Table 2 The restriction emzyme digestion system
2、FliC NC Prokaryotic expression and purification of recombinant flagellin
(1) The correct pET-28 alpha (+) -fliC will be identified by DNA sequencing NC The recombinant expression plasmid was electrotransformed into BL21 (DE 3) competent cells, which were then plated on LB solid medium of kanamycin (Kan+) and cultured overnight in a bacterial incubator at 37 ℃. Single positive clones were picked from LB plates of overnight kanamycin, added to 6mL of Kan+ containing 2 XYT medium, and grown overnight in a shaker at 37 ℃. The next day, 5mL of overnight cultured seed solution was inoculated at 1:100 into 500mL of fresh autoclaved 2 XYT medium, and 500. Mu.L (30. Mu.g/mL) of Kan+ was added and shake-cultured at 37℃to OD 600nm When the expression was =0.6 to 0.7, 5mL of 100mM IPTG was added to a final concentration of 1mM/L, the expression was continued at a constant temperature of 37℃for 4 hours, and the cells were collected by centrifugation at 12000rpm X20 min. Lysing the cells with a B-PER reagent, andNi-TED2000Packed Columns purification kit (MACHEREY-NAGEL Co., cat. No. 745120.25) was used to purify recombinant proteins, using Pierce TM High Capacity Endotoxin Removal Spin Column endotoxin removal kit (ThermoFisher Scientific, cat# 88276) removes Lipopolysaccharide (LPS) from flagellin, ensures that the LPS content is less than 0.05EU/mL, and then the purified protein concentration is 2.16mg/mL as measured by BCA method.
(2) Identification of recombinant proteins
Purified recombinant proteins were validated using SDS-PAGE and immunoblotting (Western blot).
After 12% SDS-PAGE electrophoresis is carried out on the purified recombinant protein sample, the target protein is transferred onto a PVDF membrane by a semi-dry transfer printing instrument under the conditions of 25V,1.0A and 30 min. The transferred PVDF membrane is placed in 10% skimmed milk prepared by PBST (PBS+0.5%Tween 20), slowly shaking and sealing for 3 hours at room temperature on a shaker, and transferring to 4 ℃ for overnight sealing. Against E.coli flagellin fliC H7 Polyclonal antibody (Tianjin biochip, cat# IM-EH 001-7) was used as primary antibody, diluted with 5% nonfat milk powder (1:10000), incubated with slow shaking on a shaker at room temperature for 1.5h and washed 4 times with PBST. Secondary goat anti-Rabbit IgG-HRP (ABclonal Co., ltd., cat#): AS 014), diluted with PBST (1:15000) and placed on a shaking table with slow shaking for 1h incubation. PBST was washed 4 times, 5 min/time, and PVDF film was developed by ECL luminescence method.
As shown in FIG. 3, SDS-PAGE results shows, recombinant flagellin fliC H7 And FliC NC The sizes are 60kDa and 28kDa, respectively. Western blot results show that recombinant flagellin fliC H7 Full length and anti-coliform flagellin FliC H7 Polyclonal antibodies react specifically, while FliC lacking the intermediate hypervariable region NC Cannot be identified, indicate FliC NC Does not possess the antigenicity of intact flagellin.
EXAMPLE 2 recombinant flagellin FliC NC Evaluation of immune adjuvant Effect
(1) Recombinant flagellin FliC NC In vitro detection of TLR5 receptor activity
Detection of recombinant flagellin FliC using human colorectal adenocarcinoma cells (Caco-2) capable of expressing TLR5 receptor as an in vitro cell model NC TLR5 receptor activity of the mutant. Caco-2 cells at 25cm 2 After the cell culture flask was full of cells, 1X 10 cells per well were used 7 Individual cells were seeded into 6-well cell culture plates and when cells were confluent with monolayers, purified FliC was added at a final concentration of 5 μg/mL NC And recombinant flagellin FliC H7 Caco-2 cells were stimulated and incubated at 37℃for 6h, untreated Caco-2 cells served as negative controls. Then, the mixture was centrifuged at 1200rpm at 4℃for 10min, and the cell culture supernatant was collected, and the concentrations of IL-8 and TNF-alpha in the cell supernatant were detected by using a human IL-8ELISA kit (Xinbo Biotechnology Co., ltd., product number: EHC 008.96) and a TNF-alpha ELISA kit (Xinbo Biotechnology Co., ltd., product number: EHC103 a.96), respectively. The results are shown in FIG. 4, which shows fliC NC Mutant and recombinant flagellin FliC H7 The stimulation of Caco-2 secretion of IL-8 and TNF- α levels were comparable, indicating FliC NC The mutant has good TLR5 receptor activity.
(2) Immune mouse serum anti-FaeG antibody level detection
Recombinant flagellin FliC to be prokaryotic expressed and purified H7 、FliC NC Mutant (50. Mu.g) and prokaryotic recombinant expressionMode antigen protein FaeG (Duan et al 2020,veterinary research,50 μg) and the like; freund's adjuvant group was prepared by emulsifying Freund's complete adjuvant (Sigma Co., cat# SLCJ 8308) or Freund's incomplete adjuvant (Sigma Co., cat# SLCJ 3267) with FaeG antigen protein at a ratio of 1:1; 6 female BALB/c mice, 6-8 weeks old, were immunized subcutaneously, respectively, of which 6 mice injected with 100 μl of sterile PBS at a concentration of 0.1M, PH =7.4 served as a negative control group. The specific immunization procedure is as follows: after the first immunization, the immunization was boosted every two weeks, for a total of 3 times. The eyebox of the mice is sampled weekly by a capillary glass tube, and the eyeballs of the mice are sampled after the painless cervical vertebra is sacrificed on the 14 th day after the last immunization, and the mice are placed in a refrigerator at 4 ℃ for standby after serum separation. The indirect ELISA method is used for detecting the specific IgG antibody titer of the mode antigen FaeG recombinant protein in the serum of the mice. The results are shown in FIG. 5, fliC NC The anti-FaeG antibody titer induced by the immune adjuvant group is equivalent to that of the Freund's adjuvant group and is obviously higher than that of FaeG single antigen group and FliC H7 Adjuvant group, indicating FliC NC The recombinant protein has better immune adjuvant effect.
(3) TR-PCR detection of expression of IL-4 and TNF-alpha inflammatory cytokines by spleen cells of immunized mice
Mice were sacrificed by cervical dislocation as in (2), spleens were aseptically removed and sheared with tissue, then fully ground with a 5mL syringe rubber piston head, and tissue residues were filtered using an autoclaved copper mesh. Centrifuging the filtered spleen cells at 1000rpm for 10min, and taking out cell sediment at the lower layer; then adding proper amount of 1X erythrocyte lysate into the cell sediment, standing at room temperature for 5min to lyse erythrocytes, centrifuging at 1000rpm for 10min, and taking the cell sediment. Washing the cell pellet with serum-free RPMI 1640 culture solution for 3 times; finally, the cells were fully resuspended in RPMI 1640 medium containing 1% of penicillin+0.1% of FBS and the cell suspension concentration was adjusted to 2.5X10 5 Individual cells/mL. Cell count 5X 10 5 Cell/well density 6 well cell culture plates were inoculated and placed at 37℃with 5% CO 2 Culturing in a constant temperature incubator. The FaeG recombinant protein with the concentration of 5 mug/mL is added to each well to stimulate, spleen cells are collected after 48 hours, and total RNA of the cells is extracted by adopting a TRNzol method.
The total RNA extracted was checked for efficiency of extraction and RNA quality by agarose gel electrophoresis. Reverse transcription of the extracted RNA into cDNA Using FastKing gDNADispelling RT SUperMix kit (Norwegian biosystems), according to Norwegian CorpPremix Ex Taq TM (Perfect Real Time) the instructions on the kit allow qRT-PCR amplification to detect the expression of inflammatory factor cells. The specific primer of the il-4 is il-4-F (SEQ ID NO. 6)/il-4-R (SEQ ID NO. 7), and the specific primer of the tnf-alpha is tnf-alpha-F (SEQ ID NO. 8)/tnf-alpha-R (SEQ ID NO. 9); the specific primer of inf-gamma is inf-gamma-F (SEQ ID NO. 10)/inf-gamma-R (SEQ ID NO. 11). The specificity of primer amplification was ensured by dissolution profile, and the data obtained was used 2 -△△CT The method is used for processing. Statistical differences for each set of data were analyzed statistically using Student's-test method in GraphPad Prism 5 software. The results are shown in FIG. 6, the immune adjuvant groups il-4, tnf-alpha and inf-gamma gene expression levels were significantly increased compared to the PBS control group, and FliC NC The expression level of 3 genes in the immune adjuvant group is significantly higher than that of FliC H7 Immune adjuvant group and Freund's immune adjuvant group, indicating FliC NC The flagella mutant has more excellent immunoadjuvant effect.
Claims (10)
1. A hypervariable region domain deleted bacterial flagellin, wherein the hypervariable region domain deleted bacterial flagellin is a FliC compared to a FliC having an intact domain H7 The 178 th amino acid to 497 th amino acid of the hypervariable region is deleted.
2. The bacterial flagellin deleted of the hypervariable region domain according to claim 1, wherein the amino acid sequence thereof is shown in SEQ ID No. 2.
3. A mutant gene encoding a bacterial flagellin deleted of the hypervariable region domain according to claim 1, wherein the mutant geneIs of E.coli H7 serotypefliCThe 534 th to 1491 th bases of the hypervariable region of the full-length gene are deleted.
4. A mutant gene of bacterial flagellin deleted of hypervariable region domain according to claim 3, wherein the nucleotide sequence is shown in SEQ ID No. 1.
5. An expression cassette, a recombinant vector, a recombinant cell or a recombinant bacterium, which comprises the mutant gene according to claim 3 or 4.
6. A method for the preparation of a hypervariable region domain deleted bacterial flagellin according to claim 1 or 2, comprising the steps of: will encode E.coli H7 serotypefliCThe base deletion of the 534 th to 1491 th hypervariable region of the full-length gene is cloned to a prokaryotic expression vector to construct a recombinant expression plasmid, and the recombinant expression plasmid is transformed into the escherichia coli engineering bacteria to be subjected to IPTG induction and then prokaryotic high-efficiency expression of fliC NC Recombinant protein is obtained.
7. The method of claim 6, wherein the nucleic acid encoding E.coli H7 serotypefliCThe GeneBank accession number of the full-length gene is AY249992.
8. Use of a bacterial flagellin deleted of a hypervariable region domain according to claim 1 or 2, a mutant gene according to claim 3 or 4, an expression cassette, a recombinant vector, a recombinant cell or a recombinant bacterium according to claim 5 for the preparation of a vaccine immunoadjuvant.
9. Vaccine immunoadjuvant, characterized in that it comprises a bacterial flagellin lacking a hypervariable region domain according to claim 1 or 2 or a mutant gene according to claim 3 or 4.
10. Vaccine immunoadjuvant according to claim 9, characterized in that it further comprises a pattern antigen FaeG antigen.
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