A bacterial flagellin lacking a hypervariable region structural domain and its preparation method and application

Abstract

The present invention discloses a bacterial flagellin lacking a hypervariable region structural domain and a preparation method and application thereof. The bacterial flagellin lacking a hypervariable region structural domain is a FliC H7 The amino acid at position 178 to position 497 of the hypervariable region is missing. The present invention constructs a truncated recombinant Escherichia coli flagellin FliC by deleting the middle hypervariable region of flagellin NC In vitro Caco‑2 cell model detection revealed that FliC NC With FliC H7 Similar and good TLR5 receptor activity can activate TLR5 signaling pathway; in vivo, FliC NC Recombinant flagellin can also induce high levels of IgG antibodies against the model antigen FaeG in vivo and inflammatory genes related to humoral and cellular immunity in spleen cells.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a bacterial flagellin lacking a hypervariable region structural domain, and a preparation method and application thereof. Background Art

[0002] Vaccine immunization is one of the most economical, effective and safe means to prevent and control various infectious diseases. Vaccination can effectively reduce the morbidity and mortality of diseases. Through vaccination, the body can obtain immune protection against specific pathogens, greatly reducing the risk of infection. However, since the immunogenicity of the antigen itself is weak, inactivated vaccines, protein subunit vaccines, nucleic acid vaccines, etc. must be added with immune adjuvants to enhance their immunogenicity and improve their immune protection effect. Although some traditional immune adjuvants such as aluminum hydroxide gel, surfactants, white oil adjuvants, etc. can enhance the immunogenicity of antigens to varying degrees, there are still many limitations such as limited immune pathways, insignificant immune effects, mainly inducing humoral immune responses, relatively high costs, and low safety. Therefore, it is necessary to develop a new immune adjuvant that is safe, has excellent immune adjuvant effects, and is low in cost.

[0003] In recent years, many pathogenic microorganisms or their components have been found to have excellent immune adjuvant effects. Among them, the development of Toll-like receptors (TLRs) ligands as adjuvants has received widespread attention. Bacterial flagellin is a ligand of TLR5. As a new type of immune adjuvant, it has strong immune adjuvant activity. It can not only activate the body's innate immune response system, but also induce cellular immunity and humoral immunity at the same time. Since McEwen first proved the immune adjuvant effect of flagellin, the structural basis and molecular mechanism of flagellin's immune adjuvant effect have always been one of the frontiers and hot issues in the international scientific community.

[0004] The crystal structure analysis of Salmonella flagellin by electron microscopy and X-ray diffraction showed that the complete flagellin includes four related domains: D0, D1, D2 and D3. Different domains of flagellin have different structural characteristics and play different biological functions. The D0 and D1 domains located at the NH2 and COOH ends of flagellin are highly conserved in different species and can activate TLR5 and / or NAIP5-NLRC4 signaling pathways to exert an immune adjuvant effect. The middle hypervariable region (V) contains the D2 and D3 domains, and the D3 domain has a high degree of variability due to exposure to the surface of the flagellar filament. The hypervariable region of flagellin mainly determines the serotype and autoimmunogenicity of the flagella, and is also necessary for the antigenicity of flagellin. However, the presence of the hypervariable region can not only induce the body to produce specific antibodies against itself, but the anti-flagellin antibodies induced by it may also neutralize TLR5 receptor signal transduction and reduce the immune effect of flagellin. In addition, the presence of the hypervariable region can also induce problems such as cytotoxic reactions in the host. Using structural biology to further clarify the structural characteristics of different domains of flagellin can provide important information for in-depth research on their functions.

[0005] Bacterial flagellin can activate the body's innate immune system through the TLR5 and NLRC4 signaling pathways, induce the secretion of pro-inflammatory factors (IL-1β, IL-8, IL-18, TNF-α, etc.), and thus exert an immune adjuvant effect. Extracellular flagellin can bind to and activate Toll-like receptor 5 (TLR5) on the cell surface, inducing the secretion of inflammatory factors such as IL-8 and TNF-α; intracellular flagellin binds to and activates the NAIP5-NLRC4 complex, inducing the production of IL-1β, IL-18 and cell apoptosis. In the process of infection by pathogenic microorganisms, the cytokines and chemokines induced by flagellin through the TLR5 and / or NLRC4 signaling pathways are the key to flagellin activating innate immunity and acquired immunity and thus exerting an immune adjuvant effect.

[0006] Flagellin has many advantages as a new type of molecular immune adjuvant: 1) Flagellin can not only induce humoral immunity, but also induce high levels of cellular immune response. 2) Flagellin can be combined with exogenous antigens in various forms (mixed / chimeric / fusion), and exert adjuvant effects through various immune pathways. 3) The protein structure has strong plasticity, and the insertion of exogenous antigens at the NH2 end, COOH end or hypervariable region does not affect its structure and TLR5 receptor activity. 4) Flagellin can overcome oral tolerance and efficiently induce proinflammatory cytokines on the mucosal surface. 5) In addition to being an immune adjuvant itself, flagellin also has the effect of enhancing other immune adjuvants. Although bacterial flagellin has many advantages as a new adjuvant, there is currently no commercially available or approved vaccine based on flagellin as an adjuvant. Only the recombinant fusion influenza-flagellin vaccine has entered Phase II clinical research. This is mainly because the antibodies induced by the body against flagellin itself when using complete bacterial flagellin as an adjuvant may interfere with the secondary immune response. Secondly, the safety of complete flagellin as an adjuvant also needs further evaluation. Therefore, it is imperative to develop, improve and optimize flagellin-based immune adjuvants in combination with the structural biology of bacterial flagellin. Summary of the invention

[0007] Purpose of the invention: The purpose of the present invention is to provide an Escherichia coli flagellin (FliC) with excellent adjuvant effect, safety and high efficiency and lacking the middle hypervariable region. NC ) and its preparation method and application, overcome the problems faced by flagellin as an immune adjuvant in clinical applications. By truncating the middle hypervariable region (V), only the conservative NH2-terminus and COOH-terminus related to the immune adjuvant effect are retained. The flagellin mutant (FliC) with the hypervariable region deleted NC ) not only has the TLR5 receptor activity of full-length flagellin, but also avoids the inherent antigenicity of flagellin, thereby preventing the induction of specific antibodies against flagellin itself due to the presence of the hypervariable region, which affects the body's secondary immune response or TLR5 signal crosstalk. In addition, the recombinant flagellin FliC NC After immunization injection into the body, it will not cause any significant systemic pro-inflammatory response, so it has the potential to be developed into a safer, more efficient and side-effect-free bacterial flagellin immune adjuvant.

[0008] Technical solution: In order to solve the above technical problems, the present invention provides a bacterial flagellin lacking a hypervariable region domain, wherein the bacterial flagellin lacking a hypervariable region domain is a FliC protein having a complete domain. H7 , amino acids 178 to 497 in its hypervariable region are missing.

[0009] Wherein, the amino acid sequence of the mutant protein is shown as SEQ ID NO.2.

[0010] The present invention also includes a mutant gene encoding the bacterial flagellin lacking the hypervariable region domain, wherein the mutant gene lacks bases 534 to 1491 of the hypervariable region of the full-length fliC gene of Escherichia coli H7 serotype.

[0011] Wherein, the nucleotide sequence of the mutant gene of bacterial flagellin lacking the hypervariable region domain is shown as SEQ ID NO.1.

[0012] The present invention also includes an expression cassette, a recombinant vector, a recombinant cell or a recombinant bacterium, which contains the mutant gene.

[0013] The present invention also includes a method for preparing the bacterial flagellin lacking the hypervariable region domain, comprising the following steps: deleting the bases 534-1491 of the hypervariable region encoding the full-length gene of fliC of Escherichia coli H7 serotype, cloning it into a prokaryotic expression vector to construct a recombinant expression plasmid, transforming it into an Escherichia coli engineered bacterium, and inducing it with IPTG to efficiently express FliC in the prokaryotic cell. NC The recombinant protein is obtained.

[0014] FliC of the present invention NC The recombinant protein lacks the hypervariable region associated with flagellin antigenicity and cannot be detected by rabbit anti-FliC in Western blot experiments. H7 The polyclonal antibody recognized FliC, indicating that the hypervariable region was missing. NC It does not have flagellin antigenicity, which can effectively avoid the interference of flagellin autoantibodies on the body's secondary immune response. NC Recombinant protein compared to FliC with complete domain H7 , retaining only the NH2-terminal and COOH-terminal FliC NC It has comparable TLR5 receptor stimulating activity in vitro. NC It has good adjuvant activity in vivo. NC As an immune adjuvant, mixed with the model antigen FaeG to immunize BALB / c mice can induce high titer anti-FaeG IgG antibodies.

[0015] The GeneBank accession number of the full-length fliC gene encoding Escherichia coli H7 serotype is AY249992.

[0016] The present invention also includes the use of the bacterial flagellin lacking a hypervariable region structural domain, the mutant gene, the expression cassette, the recombinant vector, the recombinant cell or the recombinant bacteria in the preparation of vaccine immune adjuvants.

[0017] The present invention also includes a vaccine immune adjuvant, which comprises the bacterial flagellin lacking the hypervariable region domain or the mutant gene.

[0018] Among them, the vaccine immune adjuvant also includes the model antigen FaeG antigen.

[0019] FliC of the present invention NC Immunization of BALB / c mice with the recombinant protein and the model antigen FaeG antigen can induce Th1 (TNF-α and IFN-γ) and Th2 (IL-4) immune responses in the spleen, with Th1 (cellular) immune responses being the main ones.

[0020] Since the middle hypervariable region was deleted, only the conserved domain of FliC was retained. NC Flagellar recombinant protein has excellent immune adjuvant effect, FliC NC Recombinant flagellin can be used as an immune adjuvant in the development of vaccines against various pathogens such as bacteria, viruses and parasites.

[0021] The preparation and application of the recombinant flagellin with the middle hypervariable region deleted of the present invention is also applicable to flagellins of other serotypes of Escherichia coli and other bacterial flagellins.

[0022] The present invention truncated the middle hypervariable region (V) of the flagellin of Escherichia coli H7 serotype, connected the conserved NH2-terminal and COOH-terminal ends with GPVD linker, and amplified FliC using SOE-PCR technology. NC , construction of truncated recombinant flagellin FliC NC .

[0023] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the FliC constructed by the present invention NC Recombinant flagellin has a similar spatial conformation and biological activity to natural flagellin; it can not only induce humoral immune responses against target antigens, but also induce cellular immune responses; it effectively improves the immune effect, innovatively improves and overcomes the bottleneck of existing bacterial flagellin as an immune adjuvant. It is expected that the conservative domain of flagellin will be used as a new molecular adjuvant in the development of clinical vaccines for bacterial, viral, parasitic and other diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 For FliC NC Schematic diagram of the construction of recombinant bacterial flagellin. N represents the NH2 end 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.

[0025] Figure 2 fliC full length and fliC NC Electrophoresis diagram of PCR amplification and identification of genes. Lane M is 2K PlusⅡDNAMarker; Lane 1 is fliC H7 Full-length gene; Lane 2 is fliC NC Gene.

[0026] Figure 3 To recombinant FliC NC SDS-PAGE and Western blot analysis of flagellin. A. Recombinant FliC NC SDS-PAGE electrophoresis of flagellin. Lane M is the protein marker; Lane 1 is FliC H7 Recombinant flagellin; Lane 2 is FliC NC Recombinant flagellin. B. Recombinant FliC NC Western blot analysis of flagellin. Lane 1 is FliC H7 Recombinant flagellin; Lane 2 is FliC NC Recombinant flagellin.

[0027] Figure 4 For FliC NC Recombinant flagellin was used to detect TLR5 receptor activity in vitro. 5 μg / mL of endotoxin-free FliC H7 and FliC NC Recombinant flagellin stimulated human colon cancer Caco-2 cells for 6 hours, and unstimulated Caco-2 cells were used as a negative control group. The cell culture supernatant was collected, and the concentrations of IL-8 and TNF-α in the cell supernatant were detected using human IL-8 and TNF-α ELISA kits, respectively.

[0028] Figure 5 Figure 1 shows the antibody level of the model antigen FaeG in the serum of immunized mice detected by ELISA. H7 、FliC NC Mice were subcutaneously immunized with recombinant flagellin and Freund's adjuvant as immune adjuvants and the model antigen FaeG (the major subunit of F4ac fimbriae adhesin of enterotoxigenic Escherichia coli). The extracted F4ac fimbriae adhesin was used as the coating antigen to detect the anti-FaeG IgG antibody titer in the serum of each group of immunized mice, among which the PBS immunization group served as the negative control group.

[0029] Figure 6The figure shows the expression of inflammatory cytokines mRNA in spleen cells of immunized mice detected by RT-PCR. RT-PCR detected the expression levels of IL-4, TNF-α and INF-γ genes in spleen cells of each group of immunized mice. A. Expression of IL-4 gene in spleen cells. B. Expression of TNF-α gene in spleen cells. C. Expression of INF-γ gene in spleen cells. DETAILED DESCRIPTION

[0030] The embodiments of the present invention will be clearly and completely described below in conjunction with the examples. Obviously, the described examples are only used to illustrate a part of the embodiments of the present invention and should not be regarded as limiting the scope of the present invention. If the specific conditions are not specified in the examples, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are regarded as conventional products that can be purchased commercially.

[0031] Example 1

[0032] 1. Cloning of Escherichia coli flagellar protein fliCNC gene and construction of recombinant plasmid

[0033] (1) FliC NC Construction and structure prediction

[0034] Modeling, prediction and analysis of FliC using the Phyre2 portal NC The secondary structure of the amino acid sequence of the flagellin mutant. Using FliC protein (UniProt accession number: A0A2Z5FPT6) as a control, the results showed that FliC NC The protein still maintains its natural and relatively independent spatial conformation.

[0035] (2) Primer design and gene amplification

[0036] According to the gene sequence encoding the flagellin fliC of enterohemorrhagic Escherichia coli published in GenBank (GeneBank accession number: AY249992), amplification primers were designed using DNASTRA software. H7 -F (SEQ ID NO. 3) and FliC H7 -R (SEQ ID NO.4) was used for PCR amplification of fliC H7 Full-length gene; Primer FliC H7 –F (SEQ ID NO.3) and FliC NC -R (SEQ ID NO.5) was used for PCR amplification of fliC NCThe reaction amplification system is shown in Table 1. The PCR reaction conditions are: 95℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 2min, 30 cycles, 72℃×10min, and 4℃ termination. The upstream and downstream primers carry BamHI-HF and SalI-HF restriction sites, respectively. The genome extracted from enterohemorrhagic Escherichia coli EHEC (O157:H7) was used as a template by boiling lysis method, and Phusion High-Fidelity DNA polymerase (Beijing Quanshijin Biological, Catalog No.: AP221-12) was used to PCR amplify fliC H7 and fliC NC Gene fragment. Results are available at Figure 2 As shown, fliC H7 Full-length gene and fliC NC The sizes of the gene fragments were approximately 1785 bp and 807 bp, respectively, which was consistent with the expected results.

[0037] Table 1 PCR amplification system

[0038]

[0039] (3) Construction of pET-28α(+)-FliCNC recombinant plasmid

[0040] fliC H7 and fliC NC The gene fragment PCR recovery and purification product and pET-28α(+) vector plasmid were double-digested in a 37°C water bath for 2h using BamHI-HF (NEB, Catalog No.: R3136) and SalI-HF (NEB, Catalog No.: R3138) endonucleases, respectively. The double-digestion reaction system is shown in Table 2. After the digestion product was purified and recovered, it was connected overnight at 16°C using T4 DNA ligase. The ligation product was transformed into DH5α competent cells and then added to LB liquid culture medium for shaking culture for 1h. An appropriate amount of culture product was coated on LB solid culture medium containing kanamycin (Kan+) and cultured overnight in a 37°C bacterial constant temperature incubator. The next day, several single colonies were picked and sent to the company for sequencing after PCR identification. Finally, DNA sequencing confirmed that the clone construction was correct, and the recombinant plasmid was named pET-28α(+)-FliC NC .

[0041] Table 2 Enzyme digestion reaction system

[0042] Table 2 The restriction emzyme digestion system

[0043]

[0044] 2. FliC NC Prokaryotic expression and purification of recombinant flagellin

[0045] (1) pET-28α(+)-FliC NC The recombinant expression plasmid was electrotransformed into BL21 (DE3) competent cells, then spread on LB solid medium containing kanamycin (Kan+), and cultured overnight in a 37°C bacterial constant temperature incubator. A single positive clone was picked from the overnight cultured kanamycin LB plate, added to 6 mL of 2×YT medium containing Kan+, and cultured overnight in a 37°C shaker. The next day, 5 mL of the overnight cultured seed solution was inoculated into 500 mL of fresh autoclaved 2×YT medium at a ratio of 1:100, and 500 μL (30 μg / mL) of Kan+ was added, and the culture was shaken at 37°C until OD 600nm =0.6-0.7, then add 5mL 100mM IPTG to make the final concentration 1mM / L, continue to induce expression at 37℃ for 4h, 4℃, 12000rpm×20min, and collect the cells by centrifugation. Use B-PER reagent to lyse the cells. The recombinant protein was purified using Ni-TED2000 Packed Columns purification kit (MACHEREY-NAGEL, Cat. No. 745120.25) and Pierce TM High Capacity Endotoxin Removal Spin Column Endotoxin Removal Kit (ThermoFisher Scientific, Cat. No. 88276) was used to remove lipopolysaccharide (LPS) from flagellar protein to ensure that the LPS content was less than 0.05 EU / ml. The concentration of the purified protein was measured by the BCA method to be 2.16 mg / mL.

[0046] (2) Identification of recombinant proteins

[0047] The purified recombinant protein was verified and analyzed by SDS-PAGE and Western blot.

[0048] After the purified recombinant protein sample was subjected to 12% SDS-PAGE electrophoresis, the target protein was transferred to the PVDF membrane using a semi-dry transfer apparatus at 25V, 1.0A, 30min. The transferred PVDF membrane was placed in 10% skim milk prepared in PBST (PBS + 0.5‰ Tween 20), and the membrane was slowly shaken on a shaker at room temperature for 3h and then transferred to 4℃ for overnight blocking. H7The polyclonal antibody (Tianjin Biochip, Catalog No.: IM-EH001-7) was used as the primary antibody, diluted with 5% skim milk powder (1:10000), incubated on a shaker at room temperature for 1.5 hours, and washed 4 times with PBST. The secondary antibody goat anti-rabbit IgG-HRP (ABclonal, Catalog No.: AS014) was diluted with PBST (1:15000) and incubated on a shaker for 1 hour. The PBST was washed 4 times, 5 minutes / time, and the PVDF membrane was developed by ECL luminescence method.

[0049] like Figure 3 , SDS-PAGE results showed that the recombinant flagellar protein FliC H7 and FliC NC The sizes were 60KDa and 28KDa respectively. Western blot results showed that the recombinant flagellar protein FliC H7 Full-length anti-E. coli flagellin FliC H7 The polyclonal antibody reacted specifically with FliC, which lacked the middle hypervariable region. NC Cannot be recognized, indicating that FliC NC It does not have the antigenicity of intact flagellin.

[0050] Example 2 Recombinant flagellin FliC NC Evaluation of immune adjuvant effects

[0051] (1) Recombinant flagellin FliC NC In vitro TLR5 receptor activity assay

[0052] Using human colorectal adenocarcinoma cells (Caco-2) expressing TLR5 receptor as an in vitro cell model, we tested the recombinant flagellin FliC. NC TLR5 receptor activity of mutants. Caco-2 cells were cultured at 25 cm 2 After the cells are full in the culture flask, 1×10 7 Cells were inoculated into 6-well cell culture plates. When the cells grew into a monolayer, purified FliC was added at a final concentration of 5 μg / mL. NC and recombinant flagellin FliC H7 Stimulate Caco-2 cells and culture at 37℃ for 6h. Untreated Caco-2 cells were used as negative control. Then centrifuge at 4℃, 1200rpm for 10min, collect cell culture supernatant, and use human IL-8 ELISA kit (Xinbosheng Biotechnology Co., Ltd., catalog number: EHC008.96) and TNF-α ELISA kit (Xinbosheng Biotechnology Co., Ltd., catalog number: EHC103a.96) to detect the concentration of IL-8 and TNF-α in the cell supernatant. The results are shown in Figure 2. Figure 4As shown, it shows FliC NC Mutant and recombinant flagellin FliC H7 Stimulating Caco-2 to secrete IL-8 and TNF-α at comparable levels suggests that FliC NC The mutant has good TLR5 receptor activity.

[0053] (2) Detection of anti-FaeG antibody levels in sera of immunized mice

[0054] Prokaryotic expression and purification of recombinant flagellin FliC H7 、FliC NC The mutant (50 μg) was mixed with the prokaryotic recombinant expressed model antigen protein FaeG (Duan et al., 2020, veterinary research, 50 μg) in equal mass; the Freund's adjuvant group fully emulsified Freund's complete adjuvant (Sigma, catalog number: SLCJ8308) or Freund's incomplete adjuvant (Sigma, catalog number: SLCJ3267) with FaeG antigen protein in a volume ratio of 1:1; 6 BALB / c female mice aged 6-8 weeks were subcutaneously immunized, of which 6 mice injected with 100 μL of sterile 0.1M PBS, pH = 7.4 were used as negative control groups. The specific immunization procedure is: after the first immunization, boost immunization is performed every two weeks, for a total of 3 immunizations. Blood was collected from the mouse eye sockets every week with a capillary glass tube. On the 14th day after the last immunization, the mice were painlessly killed by cervical vertebrae and blood was collected from the eyeballs. The serum was separated and placed in a 4°C refrigerator for later use. The indirect ELISA method was used to detect the specific IgG antibody titer against the model antigen FaeG recombinant protein in mouse serum. Figure 5 As shown, FliC NC The anti-FaeG antibody titer induced by the immune adjuvant group was comparable to that of the Freund's adjuvant group, and was significantly higher than that of the FaeG antigen alone group and the FliC H7 Adjuvant group, indicating that FliC NC Recombinant protein has better immune adjuvant effect.

[0055] (3) TR-PCR detection of IL-4 and TNF-α expression of inflammatory cytokines in spleen cells of immunized mice

[0056] The mice were killed by cervical dislocation according to the method in (2), and the spleen of the mice was removed aseptically and chopped with tissue scissors, and then fully ground with the rubber piston head of a 5mL syringe, and the tissue residue was filtered using an autoclaved copper mesh. The filtered spleen cells were centrifuged at 1000rpm for 10min, and the cell pellet at the bottom layer was removed; then an appropriate amount of 1× red blood cell lysis buffer was added to the cell pellet and the red blood cells were lysed at room temperature for 5min, and the red blood cells were centrifuged at 1000rpm for 10min, and the cell pellet was removed. The cell pellet was washed three times with serum-free RPMI 1640 culture medium; finally, the cells were fully resuspended in RPMI 1640 culture medium containing 1% penicillin-streptomycin + 0.1% FBS, and the concentration of the cell suspension was adjusted to 2.5×10 5 cells / mL. The cells were 5×10 5 The cells were inoculated at a density of 10 cells / well in a 6-well cell culture plate and cultured in a constant temperature incubator at 37°C and 5% CO2. 5 μg / mL FaeG recombinant protein was added to each well for stimulation. After 48 hours, spleen cells were collected and total cellular RNA was extracted using the TRNzol method.

[0057] The extraction efficiency and quality of the extracted total RNA were tested by agarose gel electrophoresis. The extracted RNA was reverse transcribed into cDNA using the FastKing gDNADispelling RT SUperMix Kit (Novozyme Biotechnology Co., Ltd.) according to the Novozyme Premix Ex Taq TM (Perfect Real Time) kit instructions were used for qRT-PCR amplification to detect the expression of inflammatory factor cells. The specific primers for il-4 were il-4-F (SEQ ID NO.6) / il-4-R (SEQ ID NO.7), the specific primers for tnf-α were tnf-α-F (SEQ ID NO.8) / tnf-α-R (SEQ ID NO.9); the specific primers for inf-γ were inf-γ-F (SEQ ID NO.10) / inf-γ-R (SEQ ID NO.11). The specificity of primer amplification was ensured by melting curve, and the obtained data were used for 2 -△△CT The Student's-test method in GraphPad Prism5 software was used to analyze the statistical differences of the data in each group. Figure 6 Compared with the PBS control group, the expression levels of IL-4, TNF-α and INF-γ genes in the immune adjuvant group were significantly increased, and FliC NC The expression levels of the three genes in the immune adjuvant group were significantly higher than those in FliC H7 The immune adjuvant group and the Freund's immune adjuvant group showed that FliC NCFlagella mutants have better immune adjuvant effects.

Claims

1. Application of bacterial flagellin lacking a hypervariable region structural domain in the preparation of a vaccine immune adjuvant, wherein the bacterial flagellin lacking a hypervariable region structural domain is compared to FliC with a complete structural domain H7 The amino acid positions 178 to 497 of its hypervariable region are missing, and its amino acid sequence is shown in SEQ ID NO.

2.

2. Use of a mutant gene encoding a bacterial flagellin lacking a hypervariable region domain in the preparation of a vaccine immune adjuvant, characterized in that: The mutant gene is of Escherichia coli H7 serotype fC The 534th to 1491th bases of the hypervariable region of the full-length gene are deleted, and its nucleotide sequence is shown in SEQ ID NO.1, which encodes FliC with a complete domain. H7 The amino acid 178 to the amino acid 497 of the hypervariable region are deleted, and the amino acid sequence is the bacterial flagellar protein shown in SEQ ID NO.

2.

3. Use of an expression cassette, a recombinant vector, a recombinant cell or a recombinant bacterium in the preparation of a vaccine immune adjuvant, characterized in that: The expression cassette, recombinant vector, recombinant cell or recombinant bacteria contains a mutant gene, and the mutant gene is a serotype of Escherichia coli H7. fC The 534th to 1491th bases of the hypervariable region of the full-length gene are deleted, and its nucleotide sequence is shown in SEQ ID NO.

1.

4. A vaccine immune adjuvant, characterized in that: It comprises a bacterial flagellin or mutant gene lacking a hypervariable region structural domain, wherein the bacterial flagellin lacking a hypervariable region structural domain is a FliC having a complete structural domain. H7 The amino acid at position 178 to position 497 of the hypervariable region is deleted, and the amino acid sequence is shown in SEQ ID NO.

2. The mutant gene is of Escherichia coli H7 serotype. fC The 534th to 1491th bases of the hypervariable region of the full-length gene are deleted, and its nucleotide sequence is shown in SEQ ID NO.

1.

5. The vaccine immune adjuvant according to claim 4, characterized in that It also includes the model antigen FaeG antigen.

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