Novel chimeric flagellin and application thereof

Abstract

The invention provides a novel chimeric flagellin and application thereof. The novel chimeric flagellin is formed by chimeric combination of a Senecavirus A VP2 fragment and an escherichia coli Nissle 1917 flagellin FliC through Linker (GGGGS) 3, soluble expression of the novel chimeric flagellin in an escherichia coli system is achieved, and the highest yield can reach 200 mg / L. In addition, through detection of in-vitro Caco2 and Raw264.7 cell models, the recombinant novel chimeric flagellin shows good TLR5 receptor activity, and can effectively activate a TLR5 signal channel.

Description

Technical field:

[0001] The invention belongs to the field of biotechnology, and specifically relates to the design, induction expression, purification method and in vitro biological activity verification of a novel chimeric protein of a type A Senecavirus VP2 fragment and Escherichia coli Nissle1917 flagellin. Background technology:

[0002] Seneca disease is a new infectious disease of pigs caused by Senecavirus A (SVA). Infected pigs usually show symptoms such as depression, fever, anorexia, lameness, and blisters on the hooves and snout. In recent years, the SVA epidemic has continued to increase in many regions of China, and the mortality rate of infected piglets has increased, causing great economic losses to the pig industry. At present, there is still no commercial vaccine available for the prevention and control of SVA infection, and the inactivated vaccines and live attenuated vaccines that are currently being studied have some defects, such as the reversion of attenuated vaccines, and the problems of virus escape and incomplete inactivation in the production of inactivated vaccines. Subunit vaccines have become the focus of vaccine research and development due to their high safety, long-lasting immunity, and large-scale production through engineered bacteria. Studies have found that the precursor protein P1 of SVA can be ultimately decomposed into four structural proteins VP1-4 by proteases such as 3C, among which the VP2 structural protein has better immunogenicity, which makes the SVA subunit vaccine designed with VP2 as the antigen the first choice for preventing SVA. [1] However, adjuvants are usually added during the manufacturing process of VP2 subunit vaccines to improve the vaccine effect, and the solubility of VP2 expressed alone is poor. Flagellin, as a new antibody adjuvant, has the advantage of being able to play an adjuvant role at a lower dose compared to other traditional aluminum gel emulsion adjuvants. It can effectively induce the host's humoral cellular immunity and cell-specific immune response. [2] .

[0003] As an important pathogen-associated molecular pattern (PAMPs), bacterial flagellin can induce pro-inflammatory responses and exert immune adjuvant activity by activating the body's TLR5 or NLRC4 signaling pathways. At present, the research on the adjuvant activity of Salmonella typhimurium flagellin has been widely carried out, and its immune adjuvant effect has been applied in the research of vaccines for various pathogens, including Yersinia pestis, Streptococcus mutans, Listeria monocytogenes, influenza A virus, West Nile virus and Plasmodium vivax. [3] Recent studies have also found that the flagellin of Escherichia coli Nissle 1917 has a similar crystal structure, which can induce host cells to secrete a variety of cytokines and regulate immune responses, showing good adjuvant potential and is expected to become another new vaccine design solution strategy. [4-5] .

[0004] The chimera of flagellin and antigen is the preferred strategy for constructing vaccines. Inserting foreign antigens at different positions of flagellin can not only maintain the immunomodulatory function, ensure the effective delivery of antigens and adjuvants, and improve the immune response, but also shorten the vaccine production time and reduce costs. At present, this chimera strategy has been fully studied in the application of Salmonella typhimurium flagellin, such as malarial parasite merozoite surface protein antigen, Yersinia pestis F1 antigen, human papillomavirus antigen, etc., all of which have shown good protective effects. [6] . However, there is a lack of research on the chimeric antigen of the flagellin of Escherichia coli Nissle 1917, probably because its structural analysis is not clear enough and the mechanism research is not yet complete. The chimeric strategy of using the flagellin of Salmonella typhimurium may not be effective and economical, leading to problems such as incorrect protein folding and low expression efficiency. Therefore, Deng Yongjun's research mixed the flagellin of Escherichia coli Nissle 1917 with antigens for immunization. The results showed that the flagellin of Escherichia coli Nissle1917 and its mutants can exert an adjuvant effect after mixed immunization with the ALVP27 model antigen. [5] . The present invention aims to solve the above-mentioned defects and explore the influence of different chimeric forms on the adjuvant effect of flagellin. The flagellin of Escherichia coli Nissle1917 was selected and chimeric with the VP2 fragment of SVA. During the implementation process, it was unexpectedly found that this chimeric form can solve the problem of poor solubility of VP2, and the soluble expression yield is significantly improved. Through the detection of in vitro Caco2 and Raw264.7 cell models, the recombinant new chimeric flagellin showed good TLR5 receptor activity and could effectively activate the TLR5 signaling pathway. At the same time, it can improve the immunogenicity of VP2 and enhance the overall efficacy of flagellin vaccine, which is expected to be used in the prevention of Seneca's disease.

[0005] [1]Ru Y,Hao R,Wu C,Li Y,Lu B,Liu H,Tian H,Li D,Shi Z,Luo J,Ma K,ZhangG,Liu X,Zheng H.Identification ofPotential Novel B-Cell Epitopes ofCapsidProtein VP2 in SenecavirusA.Microbiol Spectr.2023Aug 17;11(4):e0447222.doi:10.1128 / spectrum.04472-22.Epub 2023Jul 10.PMID:37428080;PMCID:PMC10433816.

[0006] [2]Hajam IA,Dar PA,Shahnawaz I,Jaume JC,Lee JH.Bacterial flagellin-apotent immunomodulatory agent.Exp Mol Med.2017Sep 1;49(9):e373.doi:10.1038 / emm.2017.172.PMID:28860663;PMCID:PMC5628280.

[0007] [3] Zhao Jing. Study on the enhancement of antibody response by flagellar protein of Salmonella typhimurium[D]. Second Military Medical University, 2011.

[0008] [4] Yang Ying, Deng Yongjun, Zhang Jin, et al. Structural analysis and epitope prediction of flagellar protein of probiotic Escherichia coli Nissle1917[J]. Chinese Journal of Biologicals, 2022, 35(06): 672-677. DOI: 10.13200 / j.cnki.cjb.003627.

[0009] [5] Deng Yongjun. Research on flagellin of probiotic Nissle1917 as a new immune adjuvant[D]. Guizhou University, 2021. DOI: 10.27047 / d.cnki.ggudu.2021.003111.

[0010] [6] Wu Wenwen. Study on the immune adjuvant activity of different domains of Escherichia coli flagellin FliC[D]. Yangzhou University, 2020. DOI: 10.27441 / d.cnki.gyzdu.2020.002019. Summary of the invention:

[0011] The first object of the present invention is to provide a novel chimeric flagellin.

[0012] The second object of the present invention is to provide a method for preparing a novel chimeric flagellin.

[0013] The third purpose of the present invention is to disclose the use of a novel chimeric flagellin in protecting animals from Seneca virus type A infection, which provides an alternative option for improving the effectiveness of Seneca virus vaccines and applicable vaccines. Specifically, it includes the following contents:

[0014] In a first aspect, the present invention provides a novel chimeric flagellin, wherein the novel chimeric flagellin is formed by chimerizing a Senecavirus type A VP2 fragment with an Escherichia coli Nissle 1917 flagellin.

[0015] Furthermore, the type A Senecavirus VP2 fragment is at the N-terminus of the Escherichia coli Nissle 1917 flagellin.

[0016] Furthermore, the novel chimeric flagellin is obtained by chimerizing a type A Senecavirus VP2 fragment with an Escherichia coli Nissle1917 flagellin via a Linker.

[0017] Preferably, the amino acid sequence of the Linker is GGGGSGGGGSGGGGS.

[0018] Preferably, the amino acid sequence of the novel chimeric flagellin is shown in SEQ ID NO: 2. The nucleotide sequence of the novel chimeric flagellin is shown in SEQ ID NO: 1.

[0019] Furthermore, the novel chimeric flagellin is used in the preparation of a vaccine for preventing Seneca virus infection.

[0020] In a second aspect, the present invention provides a method for preparing a novel chimeric flagellin, comprising the following steps:

[0021] S1: Full gene synthesis of a novel chimeric flagellin VP2-L-FliC, the nucleotide sequence of the novel chimeric flagellin is shown in SEQ ID NO: 1;

[0022] S2: The nucleotide sequence of the novel chimeric flagellin VP2-L-FliC is cloned into the expression vector pET-28a(+) through the BamHI and SalI restriction sites to construct the plasmid VP2-L-FliC in pET-28a(+), and the recombinant Escherichia coli expression vector is transformed into the competent BL21(DE3) to obtain a recombinant strain for producing the novel chimeric flagellin;

[0023] S3: Inducing the expression of the recombinant strain obtained in step S2;

[0024] S4: Separate and purify the bacterial solution obtained in step S3 to obtain a novel chimeric flagellar protein.

[0025] Furthermore, a novel chimeric flagellin is prepared by the above preparation method.

[0026] In a third aspect, the present invention provides a novel chimeric flagellin for use in preparing a vaccine for preventing Seneca virus infection.

[0027] Beneficial effects of the present invention: Compared with the prior art, the novel chimeric flagellin of the present invention exhibits the following significant advantages: 1) The solubility of the VP2 fragment expressed alone is poor. It was unexpectedly discovered that the novel chimeric flagellin of the present invention can solve the problem of VP2 fragment inclusion body expression, and the novel chimeric flagellin is basically expressed in a soluble manner. 2) The soluble expression amount of the novel chimeric flagellin can reach about 200 mg / L. 3) Compared with other chimeric proteins, the novel chimeric flagellin of the present invention has better TLR5 receptor activity effect in vitro. 4) The novel chimeric flagellin of the present invention can activate T lymphocytes, improve the immunogenicity of VP2, and enhance the overall efficacy of the flagellin vaccine. Description of the drawings:

[0028] Figure 1 : Plasmid map of the recombinant expression vector VP2-L-FliC in pET28a(+).

[0029] Figure 2 : SDS-PAGE electrophoresis of the soluble expression levels of the three chimeric proteins. Figure a is FliC Δ195-320 -L-VP2, lane 1: protein molecular weight standard; lane 2: uninduced sample; lane 3: 10℃ induced sample; lane 4: bacterial lysate precipitate; lane 5: bacterial lysate supernatant. Δ274-406 -L-VP2, lane 1: 10℃ induced sample; lane 2: bacterial lysate precipitate; lane 3: bacterial lysate supernatant. c is VP2-L-FliC, lane 1: protein molecular weight standard; lane 2: uninduced sample; lane 3: 37℃ induced sample; lane 4: bacterial lysate precipitate; lane 5: bacterial lysate supernatant. d is VP2 fragment, lane 1: bacterial lysate supernatant; lane 2: bacterial lysate precipitate.

[0030] Figure 3 : SDS-PAGE electrophoresis of the purified novel chimeric protein. Lane 1: protein molecular weight standard; Lane 2: uninduced sample; Lane 3: 37℃ induced sample; Lane 4: bacterial lysate precipitate; Lane 5: bacterial lysate supernatant; Lane 6: lysate supernatant flow-through sample; Lane 7: rinse sample; Lane 8: eluted sample 1; Lane 9: eluted sample 2; Lane 10: eluted sample 3; Lane 11: eluted sample 4.

[0031] Figure 4 : Western blot results of the purified chimeric protein.

[0032] Figure 5 :Dynamic light scattering (DLS) results of VP2-L-FliC protein

[0033] Figure 6:Transmission electron microscopy (TEM) results of VP2-L-FliC protein

[0034] Figure 7 : In vitro TLR5 receptor activity detection of the three chimeric proteins after purification. Human colon cancer Caco2 cells and mouse macrophage Raw264.7 cells were stimulated with 20 μg / mL endotoxin-free chimeric proteins for 12 hours, respectively, with unstimulated cells as negative control groups. 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 mouse TNF-α ELISA kits, respectively. Specific implementation method:

[0035] 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.

[0036] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, protein recombinant expression technology, cell culture, and conventional techniques in related fields in the technical field.

[0037] In the specific implementation process of the present invention, three different forms of chimeric flagellin were constructed to evaluate their effects on the flagellin adjuvant effect. The three chimeric flagellins are: VP2 is chimeric to the N-terminus of flagellin FliC to form VP2-L-FliC; VP2 is chimeric to the hypervariable region D3 of FliC to form FliC Δ274-406 -L-VP2; and inserting VP2 into the hypervariable region D2D3 of FliC to form FliC Δ195-320 -L-VP2.

[0038] Glossary

[0039] 1. Linker: In biochemistry and molecular biology, "Linker" or "linker peptide" refers to a short peptide sequence that connects two or more functional peptides or proteins. The design of linker peptides usually needs to consider their length, amino acid composition, and spatial conformation to ensure that they do not interfere with the function between the connected proteins while maintaining the desired biological activity.

[0040] 2. Chimerism: In biochemistry and molecular biology, "chimerism" usually refers to combining two or more biological molecules (such as proteins, DNA or RNA) from different sources to form a new molecule. For example, a chimeric protein is a protein composed of protein domains or functional domains from different sources.

[0041] Example 1 Construct and express the VP2-L-FliC chimeric protein, and optimize the recombinant expression process.

[0042] (1) Construction of recombinant plasmid

[0043] The complete amino acid sequence of the chimeric Senecavirus VP2 fragment and the Escherichia coli Nissle 1917 flagellin, as shown in SEQ ID NO.2, was provided to GeneWeiZ for codon optimization, and the VP2-L-FliC coding gene after codon optimization was sent to GeneWeiZ for synthesis. The sequence was cloned into the vector pET-28a(+) through the BamHI and SalI restriction sites to construct the plasmid VP2-L-FliC inpET-28a(+).

[0044] The construction methods of the other two chimeric protein recombinant plasmids were the same as above.

[0045] (2) Construction and identification of recombinant bacteria

[0046] The obtained recombinant plasmid VP2-L-FliC inpET-28a(+) was transformed into competent cells of Escherichia coli BL21(DE3), coated with LB plates containing kanamycin sulfate, and cultured at 37°C. A single positive clone was picked and cultured in LB liquid medium containing kanamycin sulfate. The plasmid was extracted and identified by double digestion with BamHI and SalⅠ, and then the digestion product was identified by agarose gel electrophoresis. After the plasmid was digested, a band of about 2690bp was visible. The Escherichia coli successfully introduced with the recombinant plasmid VP2-L-FliCin pET-28a(+) was named VP2-L-FliC in pET-28a(+) / BL21(DE3). The construction and identification methods of the other two chimeric protein recombinant bacteria were the same as above.

[0047] The empty plasmid pET-28a(+) was transformed into Escherichia coli BL21(DE3) competent cells to obtain the negative control strain pET-28a(+) / BL21(DE3).

[0048] (3) Inducible expression of chimeric protein

[0049] Induce the recombinant bacteria VP2-L-FliC inpET-28a(+) / BL21(DE3) to express the chimeric protein VP2-L-FliC, and the negative control is the control strain pET-28a(+) / BL21(DE3). The specific method is as follows:

[0050] ① Pick a positive single colony of the recombinant bacteria, inoculate it into LB liquid medium (containing 10g / L tryptone, 5g / L yeast powder, 10g / L NaCl) containing 80μg / mL kanamycin sulfate, and culture it at 37℃ overnight.

[0051] ② Take 300 μl of the recombinant bacterial culture medium obtained in step ① and inoculate it into 300 mL of fresh LB liquid culture medium containing 80 μg / mL kanamycin sulfate, and culture it at 37°C for 3-4 hours until the OD600 reaches 0.6-0.8.

[0052] ③Add IPTG with a final concentration of 0.8mmol / L to the bacterial solution with an OD600 of 0.6-0.8, and continue to culture at 37℃ for 6h. Compared with the control strain, the genetically engineered recombinant strain VP2-L-FliC in pET-28a(+) / BL21(DE3) showed a target protein band at about 90kDa, which was consistent with the theoretical value, and the expression level could reach 200mg / L.

[0053] The induction conditions for the other two chimeric proteins were 10°C, a final IPTG concentration of 0.01 mmol / L, and induction for 32 h.

[0054] (4) Purification of chimeric protein

[0055] ① The induced recombinant bacteria VP2-L-FliC in pET-28a(+) / BL21(DE3) and the control strain pET-28a(+) / BL21(DE3) were centrifuged at 4°C, 12000 rpm for 5 min, the bacteria were collected, and the bacteria were washed twice with PBS buffer.

[0056] ② Resuspend the cells in Binding buffer (20mM PB pH7.4 + 500mM NaCl + 75mM imidazole + 0.1mM PMSF + 0.05mM DTT), place the cell suspension in an ice bath, and lyse the cells with ultrasound at a power of 60W for 3s and a pause of 3s until the cell solution is clear to obtain the lysate of the recombinant bacteria. The new chimeric protein is basically expressed in a soluble form.

[0057] ③ The lysate of the recombinant bacteria was centrifuged at 4°C, 10,000 rpm for 20 min, and the supernatant of the lysate of the recombinant bacteria was collected. The chimeric protein VP2-L-FliC was purified using Ni NTABeads 6FF column (Tian Di Ren He, catalog number: SA005025), and gradient elution was performed using Elutionbuffer (20 mM PB pH 7.4 + 500 mM NaCl + 100-500 mM imidazole).

[0058] ④SDS-PAGE electrophoresis was used to detect the purification effect of the chimeric protein VP2-L-FliC and to determine the concentration of the purified protein. Figure 3 As shown, a band of about 90 kDa appeared in lanes 8-11 of the purified chimeric protein, and the purity reached more than 85%.

[0059] The purification methods of the other two chimeric proteins were the same as above.

[0060] (5) Western blot analysis of chimeric proteins

[0061] The purified chimeric protein sample was subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane at a constant current of 300 mA for 90 min. The transferred PVDF membrane was placed in 10% skimmed milk powder prepared in TBST (TBS + 0.5% Tween 20) and blocked by slowly shaking on a shaker at room temperature for 2 h. Anti-His-tag mouse monoclonal antibody (Biyuntian, catalog number: AF2873) was used as the primary antibody, diluted with antibody diluent (1:2000), incubated on a shaker at room temperature for 2 h, and washed 3 times with TBST, 5 min / time, and the PVDF membrane was developed using the ECL luminescence method. Western blot results are shown in the figure. Figure 4 As shown, the chimeric protein VP2-L-FliC can react specifically with anti-His-tag mouse monoclonal antibody, and the protein size is about 90 kDa.

[0062] The Western blot analysis method for the other two chimeric proteins was the same as above.

[0063] Example 2 Structural confirmation analysis of chimeric protein VP2-L-FliC

[0064] (1) The chimeric protein VP2-L-FliC purified in Example 1 was concentrated five times and then replaced with a buffer of 20 mm PB pH 7.4. After overnight assembly at 4°C for 24 hours, the particle size of the VP2-L-FliC protein of the present invention was measured by dynamic light scattering (DLS) (Malvern particle size analyzer). The results are as follows: Figure 5As shown, dynamic light scattering (DLS) results show that the particle size of VP2-L-FliC protein after assembly is 10-30nm, which is close to the theoretical value in existing literature.

[0065] (2) The morphology and size of the VP2-L-FliC protein were detected by TEM (Talos F200C). 5 μL of the VP2-L-FliC protein of the present invention was added dropwise to a copper mesh for adsorption for 1 min. The adsorption was performed with filter paper until a thin water film was formed. 10 μL of

[0066] 2% phosphotungstic acid negative staining solution, stain for 1 minute, filter paper absorbs excess dye, and after drying, observe through transmission electron microscope (TEM). The results are as follows Figure 6 As shown, the morphology and size of the particles formed by self-assembly of VP2-L-FliC protein were consistent with the results of dynamic light scattering (DLS).

[0067] Example 3 Detection of TLR5 receptor activity of chimeric protein VP2-L-FliC in vitro

[0068] (1) The chimeric protein purified in Example 1 was concentrated five times and then the buffer was replaced with PBS. The lipopolysaccharide (LPS) in the flagellin was removed using the ToxinEraserTMEndotoxin Removal Kit (GenScript, Catalog No.: L00338). The LPS content was ensured to be less than 0.1EU / mL using the ToxinSensorTMGel Clot Endotoxin Assay Kit (GenScript, Catalog No.: L00351). The protein concentration after endotoxin removal was measured by the BCA method and was 800mg / L.

[0069] (2) Human colorectal adenocarcinoma cells (Caco2) expressing TLR5 receptor were used as an in vitro cell model to detect the TLR5 receptor activity of the chimeric protein. Caco2 cells were grown at 10 cm 2 After the cells are grown in the culture dish, 3 × 10 5 The cells were inoculated into a 12-well cell culture plate and cultured overnight at 37°C and 5% CO2. The purified chimeric protein was added to stimulate Caco2 cells for 12 hours at a final concentration of 20 μg / mL. Untreated Caco2 cells were used as negative controls. The cell culture supernatant was collected and centrifuged at 4°C and 3000 rpm for 20 minutes. The concentration of IL-8 in the cell supernatant was detected using a human IL-8 ELISA kit (ELISA, catalog number: MM-1558H1). The results are shown in Figure 2. Figure 7 As shown in a, VP2-L-FliC chimeric protein significantly induced Caco2 cells to secrete IL-8, and its level was higher than that of FliC Δ274-406 -L-VP2, while with FliCΔ195-320 This indicates that VP2-L-FliC has good TLR5 receptor activity and its activity is higher than that of FliC. Δ274-406 -L-VP2.

[0070] (3) Next, mouse macrophages (Raw264.7) that can express TLR5 receptors were used as an in vitro cell model to further detect the TLR5 receptor activity of the chimeric protein. The experimental method also followed the processing flow of Caco2 cells, but a mouse TNF-α ELISA kit (ELISA, catalog number: MM-0132M2) was used. The ELISA results are shown in Figure 2. Figure 7 As shown in b, VP2-L-FliC significantly stimulated Raw264.7 cells to secrete TNF-α, and FliC Δ195-320 -L-VP2 had a significantly higher induction level than FliC Δ274-406 -L-VP2, further confirming the good TLR5 receptor activity of VP2-L-FliC.

[0071] Although VP2-L-FliC and FliC Δ195-320 -L-VP2 showed similar performance in TLR5 receptor activity, but in order to achieve the same adjuvant effect, FliC Δ195-320 -L-VP2 is significantly more difficult to prepare than VP2-L-FliC. Δ195-320 The soluble expression of -L-VP2 is only one tenth of that of VP2-L-FliC, and its preparation process needs to be induced at low temperature, and the whole process is time-consuming and complicated. Therefore, the present invention preferentially selects VP2-L-FliC as a new chimeric protein to improve the feasibility of research and application.

Claims

1. A novel chimeric flagellin, characterized in that: The novel chimeric flagellin is formed by chimerizing a type A Senecavirus VP2 fragment with an Escherichia coli Nissle 1917 flagellin.

2. The novel chimeric flagellin according to claim 1, characterized in that The type A Senecavirus VP2 fragment is at the N-terminus of the Escherichia coli Nissle 1917 flagellin.

3. The novel chimeric flagellin according to claim 2, characterized in that The novel chimeric flagellin is obtained by chimerizing a type A Senecavirus VP2 fragment with an Escherichia coli Nissle 1917 flagellin via a Linker.

4. The novel chimeric flagellin according to claim 3, characterized in that The amino acid sequence of the Linker is GGGGSGGGGSGGGGS.

5. The novel chimeric flagellin according to claim 1, characterized in that The amino acid sequence of the novel chimeric flagellin is shown in SEQ ID NO:

2.

6. The novel chimeric flagellin according to claim 1, characterized in that The nucleotide sequence of the novel chimeric flagellin is shown in SEQ ID NO:

1.

7. Use of the novel chimeric flagellin according to claims 1-6 in the preparation of a vaccine for preventing Seneca virus infection.

8. A method for preparing a novel chimeric flagellin, comprising the following steps: S1: Full gene synthesis of a novel chimeric flagellin VP2-L-FliC, the nucleotide sequence of the novel chimeric flagellin is shown in SEQ ID NO: 1; S2: The nucleotide sequence of the novel chimeric flagellin VP2-L-FliC is cloned into the expression vector pET-28a(+) through the BamHI and SalI restriction sites to construct the plasmid VP2-L-FliC in pET-28a(+), and the recombinant Escherichia coli expression vector is transformed into the competent BL21(DE3) to obtain a recombinant strain for producing the novel chimeric flagellin; S3: Induce the expression of the recombinant strain obtained in step S2; S4: Separate and purify the bacterial solution obtained in step S3 to obtain a novel chimeric flagellar protein.

9. A novel chimeric flagellin prepared by the preparation method according to claim 8.

10. Use of the novel chimeric flagellin according to claim 9 in the preparation of a vaccine for preventing Seneca virus infection.

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