Porcine transmissible gastroenteritis virus epitope vaccine as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a swine transmissible gastroenteritis virus epitope vaccine and a preparation method and application thereof, the amino acid sequence of FliCS.T-TGEV-S fusion protein is shown as SEQ ID NO.3, and the nucleotide sequence of the FliCS.T-TGEV-S fusion protein is shown as SEQ ID NO.4. The fusion protein FliCS.T-TGEV-S prepared by the invention is used as an epitope vaccine based on dominant antigen epitopes of TGEV, and combines advanced means such as immunodominant epitopes, a fusion protein technology and genetic engineering, so that the specificity and the protection effect of the vaccine are enhanced. The vaccine not only can induce high-level antibody response and effectively inhibit TGEV infection, but also has relatively high safety and stability. Compared with a traditional vaccine, the vaccine has remarkable advantages in the aspects of production cost, immune efficacy, protection durability and the like, and a feasible TGE prevention and control scheme is provided for the pig industry.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a porcine transmissible gastroenteritis virus epitope vaccine and a preparation method and application thereof. Background Art

[0002] Porcine transmissible gastroenteritis virus (TGEV) is a coronavirus that causes transmissible gastroenteritis (TGE) in swine, an acute, highly contact gastroenteritis. The disease is particularly harmful to young pigs, especially piglets under 1 week old, where the mortality rate can be as high as 100%, causing severe diarrhea and dehydration, resulting in huge economic losses and burdens on the breeding industry. With the scale of the pig industry, the risk of TGE transmission has increased significantly, and it is particularly necessary to strengthen prevention and control. At present, the prevention and control strategies of TGEV mainly include vaccination, optimization of breeding management, and environmental disinfection. However, the prevention and control effect of existing vaccines is not ideal, so the development of more efficient and safe vaccines is of great significance to the pig industry.

[0003] TGEV belongs to the Coronavirus family and is an enveloped single-stranded positive-sense RNA virus composed of S, M, E and N proteins. The S protein (Spike) is the main antigen of TGEV and is responsible for the binding of the virus to host cells. In particular, its receptor binding domain (RBD) can bind to the receptors of pig intestinal epithelial cells, becoming the key to viral invasion. Studies have shown that viral infection can be effectively inhibited by blocking the binding of the S protein to host cells. Therefore, the RBD of the S protein is considered to be an ideal target for the development of TGEV vaccines. After infection, TGEV rapidly replicates in intestinal epithelial cells, causing acute enteritis and mucosal damage. Because the piglet immune system is not yet mature, diarrhea, dehydration and electrolyte imbalance caused by TGEV infection are particularly fatal in piglets. In addition, TGEV infection reduces the growth efficiency and feed conversion rate of pig herds, increasing the burden of breeding.

[0004] The vaccines currently used for TGEV prevention and control mainly include inactivated vaccines, attenuated vaccines and genetically engineered subunit vaccines. Inactivated vaccines are prepared by killing the whole TGEV virus by chemical or physical methods. Although relatively safe, they induce weak immune responses, have limited immune protection effects, and require multiple vaccinations to maintain a longer protection period. Attenuated vaccines are prepared from attenuated TGEV, which can replicate to a limited extent in the body and induce a more persistent immune response. However, since attenuated vaccines have a certain degree of residual virulence, there are potential safety hazards, especially in piglets with weak immunity, which may cause mild infections. In addition, the production cycle of attenuated vaccines is long and quality control is difficult. Genetically engineered subunit vaccines construct vaccine components by expressing key viral antigen proteins or their antigenic epitopes, which are relatively safe, and can induce specific immune responses against TGEV through the precise selection of antigenic epitopes. However, at present, such vaccines still have deficiencies in immune efficacy and broad spectrum, and their antibody titers are usually low, making it difficult to provide adequate protection. In addition, the production process of subunit vaccines is complex and costly. Although existing vaccines have reduced the spread of TGE to some extent, they still face problems such as limited effectiveness, poor immune persistence, and high costs. These limitations have prompted researchers to continue exploring new, efficient, and safe TGEV vaccines.

[0005] Epitope vaccines are an emerging vaccine technology that is mainly based on identifying the most immunogenic antigenic epitopes in pathogens and inducing highly specific immune responses through targeted immunity. This vaccine development strategy is expected to make up for the shortcomings of traditional vaccines and significantly improve the safety and immune efficacy of vaccines. The advantages of epitope vaccines are mainly reflected in the following points: (1) Strong targeting: epitope vaccines can accurately activate the host immune system by selecting viral antigen epitopes and avoid non-specific reactions caused by non-critical antigens. This makes the design of epitope vaccines more targeted and reduces the risk of adverse reactions. (2) Enhanced immunogenicity: epitope vaccines use the immunodominant B cell antigen epitopes in TGEV S protein to induce high levels of neutralizing antibodies, block the virus's receptor binding process, and thus effectively inhibit viral infection. Compared with traditional vaccines, epitope vaccines have more significant immune efficacy. (3) Simplified production process: The construction of epitope vaccines through genetic engineering can improve the controllability of the production process, shorten the production cycle, and reduce production costs. This makes epitope vaccines more marketable and especially suitable for large-scale promotion and application. Epitope vaccine technology has shown good application prospects in many fields and has proven its superiority in controlling viral infection in studies. Therefore, applying epitope vaccine technology to the development of TGEV vaccines may provide a more ideal solution for the prevention and control of TGE in pigs. Summary of the invention

[0006] The purpose of the present invention is to provide a porcine transmissible gastroenteritis virus epitope vaccine and a preparation method and application thereof. The epitope vaccine is derived from the dominant B cell antigen epitope in the RBD region of the TGEV S protein, which solves the problem that the existing vaccine is not effective, and through a unique fusion protein design and autologous adjuvant effect, the immune effect and safety of the vaccine are greatly improved.

[0007] The objective of the present invention is achieved through the following technical solutions: The present invention provides a FliC S.T -TGEV-S fusion protein, the FliC S.T The amino acid sequence of the -TGEV-S fusion protein is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4.

[0008] Furthermore, the FliC S.T -TGEV-S fusion protein has an amino acid sequence as shown in SEQ ID NO.5 and a nucleotide sequence as shown in SEQ ID NO.6. S.T As a skeleton protein; replace FliC with the dominant B cell antigen epitope in the RBD region of the TGEV S protein with the amino acid sequence as shown in SEQ ID NO.1 and the nucleotide sequence as shown in SEQ ID NO.2 S.T Construction of B cell antigen epitopes with low immunogenicity and exposed on the protein surface.

[0009] The present invention also provides a FliC S.T -The method for constructing TGEV-S fusion protein specifically comprises the following steps: (1) Synthetic plasmid pUC57-FliC S.T -TGEV-S was used as the amplification template for PCR amplification to obtain F S.T - TGEV-S Target gene fragment; (2) Combine pET-28α(+) plasmid and purified F S.T -TGEV-S The target gene fragment was subjected to double restriction digestion reaction, and then the purified target gene fragment was mixed with pET-28α(+) plasmid product and T4 DNA ligase to obtain a ligation product, which was then transformed into DH5α competent cells for culture to obtain the recombinant plasmid pET-28α-FliC S.T -TGEV-S; (3) The recombinant expression plasmid pET28α-FliC S.T-TGEV-S was transferred into Escherichia coli for culture, induced for expression, and then ultrasonically broken and centrifuged for purification to obtain the FliC S.T -TGEV-S fusion protein.

[0010] Furthermore, in step (1), the primer sequences used in the PCR amplification process are shown in SEQ ID NO.7-8.

[0011] Furthermore, in step (2), the double enzyme digestion reaction system is configured as follows: 60 μL of the target gene fragment or the plasmid, 2 μL each of BamHI and SacI, 10 μL of 10×CutSmart Buffer, and 26 μL of ddH2O. The above reagents are added to a centrifuge tube in sequence, mixed thoroughly, and reacted in a 37°C constant temperature water bath for 3 hours.

[0012] The present invention also provides a porcine transmissible gastroenteritis virus epitope vaccine, the porcine transmissible gastroenteritis virus epitope vaccine comprises the FliC S.T -TGEV-S fusion protein.

[0013] The present invention also provides a FliC S.T -Application of TGEV-S fusion protein in the preparation of drugs for preventing and treating porcine transmissible gastroenteritis.

[0014] Furthermore, the medicine includes a vaccine or an antibody.

[0015] Furthermore, the FliC S.T -TGEV-S fusion protein can induce the production of neutralizing antibodies against TGEV after being used to immunize mice.

[0016] Furthermore, the FliC S.T -TGEV-S fusion protein can be detected by anti-FliC S.T and recognized by polyclonal antibodies against TGEV S protein.

[0017] The FliC provided by the present invention S.T -Beneficial effects of TGEV-S fusion protein: (1) Auto-adjuvant effect: Using the Salmonella typhimurium flagellin (FliC) with adjuvant effect as the skeleton, the functional fusion expression of the dominant B cell antigen epitope in the RBD region of the TGEV S protein and FliC was achieved for the first time. As an immunostimulatory molecule, FliC can significantly activate the host's innate and adaptive immune responses, so that the fusion protein vaccine can stimulate cellular immune responses while inducing the production of neutralizing antibodies. By adding the FliC skeleton to the fusion protein vaccine, its immune protection effect against TGEV can be significantly enhanced. The fusion protein can achieve significant immune effects without the need for additional adjuvants, reducing production costs and simplifying immunization operations.

[0018] (2) Enhanced immune protection effect: The fusion protein of the present invention can not only induce efficient humoral immunity, but also stimulate cellular immunity, thereby enhancing the protective effect of the vaccine. The fusion protein can effectively induce neutralizing antibodies against TGEV and enhance cellular immune response, providing comprehensive immune protection for the piglet population.

[0019] (3) Excellent production characteristics: The production process of the fusion protein is simple and low-cost, and is suitable for large-scale promotion and application. Compared with the high cost and complex process of traditional vaccines, the vaccine of the present invention has good production efficiency and significantly saves manpower and resources.

[0020] In summary, the fusion protein FliCS.T-TGEV-S prepared by the present invention is an epitope vaccine based on the dominant antigenic epitope of TGEV, which combines advanced means such as immune dominant epitopes, fusion protein technology and genetic engineering to enhance the specificity and protective effect of the vaccine. This vaccine can not only induce a high level of antibody response and effectively inhibit TGEV infection, but also has high safety and stability. Compared with traditional vaccines, the vaccine of the present invention has significant advantages in production cost, immune efficacy, and protection durability, and provides a practical TGE prevention and control solution for the pig industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 Schematic diagram of the construction of F18-PEDV-S chimeric pilus, where E represents the dominant neutralizing B cell antigen epitope in the RBD region of the PEDV S protein.

[0023] Figure 2It is the PCR amplification result of FliCS.T-TGEV-S chimeric gene, where lane M is 2K Plus Ⅱ DNA Marker; lane 1 is FliCS.T-TGEV-S chimeric gene.

[0024] Figure 3 This is the electrophoresis diagram of PCR identification of the FliCS.T-TGEV-S recombinant strain, where lane M is 2K Plus Ⅱ DNA Marker; lanes 1-4 are the PCR amplification results of different clones of the FliCS.T-TGEV-S recombinant strain.

[0025] Figure 4 It is the SDS-PAGE diagram of the purified FliCS.T-TGEV-S fusion protein, FliCS.T and TGEV-S proteins, where M is the protein molecular mass standard; lane 1 is the purified FliCS.T-TGEV-S fusion protein; lane 2 is the purified FliCS.T recombinant protein; lane 3 is the purified TGEV-S recombinant protein.

[0026] Figure 5 Western blot images of purified FliCS.T-TGEV-S fusion protein, FliCS.T and TGEV-S proteins.

[0027] Figure 6 ELISA was used to detect the level of specific anti-TGEV-S IgG antibodies in the serum of mice immunized with FliCS.T-TGEV-S fusion protein. Note: * indicates P < 0.05, ** indicates P < 0.01.

[0028] Figure 7 ELISA was used to detect the expression levels of inflammatory cytokines IL-4, IL-6, IFN-γ and TNF-α in the spleen cells of immunized mice.

[0029] Figure 8 Neutralizing antibody titer of immunized mouse serum against TGEV (SHXB) virus. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Unless otherwise specified, the chemical reagents, biochemical reagents and materials used in the present invention can be obtained from commercial sources.

[0036] Example 1 FliC S.T -Construction, expression and identification of TGEV-S fusion gene (1) FliC S.T -Construction and structure prediction of TGEV-S fusion protein The B cell epitope prediction of the RBD region of TGEV S protein was performed by using the bioinformatics online tool IEDB (http: / / www.iedb.org / ). S.T -Schematic diagram of the construction of TGEV-S fusion protein Figure 1 Specifically, FliC S.T(bacterial flagellin protein) (its amino acid sequence is shown in SEQ ID NO.5, and its gene sequence is shown in SEQ ID NO.6) as the backbone protein, and the dominant B cell antigen epitope in the RBD region of TGEV S protein (its amino acid sequence is shown in SEQ ID NO.1, and its gene sequence is shown in SEQ ID NO.2) is substituted for FliC S.T The B cell antigen epitope with low immunogenicity and exposed on the protein surface was constructed to obtain FliC S.T -TGEV-S fusion protein (its amino acid sequence is shown in SEQ ID NO.3, and its gene sequence is shown in SEQ ID NO.4).

[0037] Subsequently, the Phyre2 portal was used to perform structural modeling of the fusion protein, and the constructed model was scored based on key parameters such as credibility, linear coverage, and homology (>90%) to screen out the FliC with the highest score. S.T -TGEV-S protein model was used as the final model. The results showed that the inserted exogenous B cell antigen epitope did not disrupt FliC S.T The native conformation of the scaffold protein and the epitope is located in FliC S.T -The surface of TGEV-S fusion protein helps to achieve the desired antigen presentation effect.

[0038] (2) F S.T -TGEV-S PCR amplification of chimeric genes Synthetic plasmid pUC57-FliC S.T -TGEV-S was used as the amplification template, and primers P1 (SEQ ID NO.7) and P2 (SEQ ID NO.8) were used for PCR amplification, and the F S.T -TGEV-S Chimeric gene fragments.

[0039] The PCR reaction system is configured as follows: pUC57-FliC S.T -TGEV-S template 4 μL, 5×Pfu enzyme buffer 10 μL, Pfu enzyme 1 μL, 2.5mM dNTP mixture 4 μL, ddH2O 27 μL, upstream and downstream primers 2 μL each, total reaction volume 50 μL.

[0040] The PCR reaction conditions were set as follows: 95°C pre-denaturation for 5 minutes, followed by 31 cycles, including 94°C denaturation for 30 seconds, 54°C annealing for 30 seconds, 72°C extension for 2 minutes, and finally 72°C extension for 7 minutes and storage at 4°C. It is worth noting that primers P1 and P2 contain restriction sites for BamHI and SacI, respectively, for subsequent cloning operations.

[0041] PCR amplification results are shown in Figure 2 ,Depend on Figure 2 It can be seen that the target band can be seen at 1536 bp, which is consistent with the expected result. The target fragment was purified and recovered using the DNA gel recovery kit (Cat. No.: DP214-03) of Tiangen Biochemical Technology Co., Ltd., providing high-quality DNA templates for subsequent experiments.

[0042] (3) pET-28α-FliC S.T -Construction of TGEV-S recombinant plasmid First, pET-28α(+) plasmid and purified F S.T -TGEV-S The target gene fragment was double-digested with BamHI (NEB, Catalog No.: R3136V) and SacI (NEB, Catalog No.: R3156V). The enzyme digestion reaction system was configured as follows: 60 μL of gene fragment or plasmid, 2 μL of BamHI and SacI, 10 μL of 10×CutSmart Buffer, and 26 μL of ddH2O. The above reagents were added to the centrifuge tube in sequence, mixed thoroughly, and reacted in a 37°C constant temperature water bath for 3 hours.

[0043] After enzyme digestion, the purified target gene fragment was mixed with the pET-28α(+) plasmid product and T4 DNA ligase, and the ligation reaction was set to proceed at 16°C overnight.

[0044] The ligation reaction system is as follows: 3 μL of pET-28α(+) plasmid digestion product, F S.T -TGEV-S 5 μL of target gene digestion product, 1 μL of T4 ligase, 2 μL of 10×T4 ligase Buffer, total volume 10 μL.

[0045] The ligation product was then transformed into DH5α competent cells, and 800 μL of non-resistant LB liquid medium was added, and cultured at 37°C and 220 rpm for 1 hour. An appropriate amount of bacterial liquid was spread on a LB solid medium plate containing kanamycin sulfate, and cultured at 37°C for 16 hours. The next day, several single colonies were picked from the plate and transferred to 5 mL of LB liquid medium containing kanamycin sulfate for expansion culture.

[0046] Using bacterial solution as template, preliminary identification was performed by PCR. The results are as follows Figure 3As shown in the figure, the four clones picked all showed the target band at about 1536 bp, which was consistent with expectations. After PCR identification and confirmation of positive clones, the samples were sent to the company for DNA sequencing. The final sequencing results verified the accuracy of the clone construction, and the successfully constructed recombinant plasmid was named pET-28α-FliC S.T -TGEV-S.

[0047] (4) FliC S.T -Expression and identification of TGEV-S fusion protein The recombinant expression plasmid pET28α-FliC S.T -TGEV-S was transformed into Escherichia coli BL21 (DE3), and a single clone was picked and inoculated into 5 mL LB medium containing kanamycin sulfate, and cultured at 37°C and 220 rpm for 12 hours. Then, PCR identification was performed using primers P1 (SEQ ID NO.7) and P2 (SEQ ID NO.8). After confirming that the clone was correct, FliC S.T -TGEV-S recombinant expression strain. Next, the overnight culture of the recombinant strain was inoculated into 500 mL of LB medium containing kanamycin sulfate at a ratio of 1:100, and cultured with shaking at 37°C until the OD600 value reached about 0.6. IPTG was then added at a final concentration of 1 mmol / L to induce expression for 4 hours. The induced bacteria were collected, broken by ultrasonication, and the precipitate was collected after centrifugation and dissolved overnight in inclusion body dissolution solution. After centrifugation again, the supernatant was taken and the protein was purified by nickel ion affinity chromatography. The purified sample was detected by SDS-PAGE electrophoresis, and the results were as follows Figure 4 As shown in Figure 2, a clear band was found at about 54 kDa. After determining the protein concentration, the purified FliC S.T -TGEV-SS fusion protein was stored at -70°C until use.

[0048] For further analysis, the purified FliC S.T -TGEV-S fusion protein samples were loaded on 12% separation gel for SDS-PAGE, and the proteins were transferred to PVDF membranes after electrophoresis. The membrane was blocked in 5% skim milk powder solution at room temperature for 1 hour, and then incubated overnight at 4°C with 1:3000 dilution of anti-TGEV S protein polyclonal antibody and 1:6000 dilution of anti-Salmonella typhimurium flagellin polyclonal antibody (primary antibodies, both homemade in this laboratory). The next day, wash with PBST 5 times for 8 minutes each, then add 1:5000 dilution of goat anti-mouse IgG-HRP secondary antibody (ABclonal, catalog number: AS003), incubate at 37°C for 90 minutes, wash again with PBST 5 times for 8 minutes each, and finally use ECL color development kit for color development. Figure 5 As shown, FliCS.T -TGEV-S fusion protein can be recognized by polyclonal antibodies against TGEV S protein and Salmonella typhimurium flagella protein, indicating that the expression of this fusion protein retains FliC S.T and biological activities of B cell antigen epitopes of TGEV S protein.

[0049] Example 2 FliC S.T -Analysis of immunogenicity of TGEV-S fusion protein (1) Mouse immunization Fifteen 6-week-old BALB / c male mice were randomly divided into three groups, with 5 mice in each group. The first group of mice was subcutaneously injected with 50 μg of FliC S.T -TGEV-S fusion protein; the second group of mice were subcutaneously immunized with the recombinantly expressed TGEV S protein mixed with an equal volume of Freund's complete adjuvant, and the immunization dose was 50 μg / mouse; the third group was used as the control group, and 100 μL of autoclaved 0.01 M PBS buffer (pH=7.4) was subcutaneously injected. All mice were immunized three times. After the first immunization, booster immunization was performed every two weeks, and incomplete Freund's adjuvant was used for the second and third immunizations in the third group. Blood was collected from the retro-orbital vein before immunization (0 days) and on days 7, 14, 21, 28, 35, and 42 after the first immunization, and serum was collected and stored at -20°C. 14 days after the last immunization (i.e., the third immunization), the mice were killed by painless cervical dislocation, and spleen cells were isolated for the detection of inflammatory cytokines.

[0050] (2) Anti-TGEV-S specific IgG antibody detection The recombinantly expressed TGEV S protein in our laboratory was used as the coating antigen and coated on a 96-well ELISA plate at a concentration of 500 ng / well. After incubation at 37°C for 1 hour, the plate was transferred to 4°C overnight. The next day, the plate was washed 3 times with PBST and blocked with 10% skim milk powder solution at 37°C for 1 hour. After discarding the blocking solution, the plate was washed 3 times with PBST, and then the post-immune serum was added with a 2-fold serial dilution (1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800), 100 μL was added to each well, and incubated at 37°C for 90 minutes. The plate was washed 3 times with PBST again, and then a 1:5000 dilution of goat anti-mouse IgG-HRP (secondary antibody, ABclonal, catalog number: AS003) was added, 100 μL per well, and incubated at 37°C for 90 minutes. After washing three times, TMB colorimetric reagent was added, color was developed at 37°C for 30 minutes in the dark, and the OD650nm value of each well was immediately read using an ELISA reader. Figure 6 As shown, FliC S.T-TGEV-S fusion protein immunization group and TGEV S protein and Freund's adjuvant immunization group were able to induce high levels of anti-TGEV S protein specific IgG antibodies from day 7, while no corresponding antibodies were detected in the PBS control group. This result shows that FliC S.T -TGEV-S fusion protein has good immunogenicity.

[0051] (3) Detection of cytokines in spleen cell culture supernatant 14 days after the last immunization, mice were killed by painless cervical dislocation, and the spleen was removed aseptically and placed in a culture dish containing PBS. The spleen was ground using a syringe piston and filtered through a 200-mesh screen to collect the cell suspension. The suspension was centrifuged at 1500 rpm for 10 minutes, the supernatant was discarded, and the red blood cells were removed using red blood cell lysis buffer and the cells were washed. Finally, the cells were resuspended in RPMI 1640 medium containing 1% penicillin-streptomycin and 1% fetal bovine serum (FBS) and the cell concentration was adjusted to 2.5×10 6 / mL. Then, 200 μL of cell suspension was inoculated into a 6-well cell culture plate, and 5 μg / mL of the corresponding protein was added to each well to stimulate the spleen cells, and cultured in an incubator at 37°C and 5% CO2 for 72 hours. After the culture, the supernatant was collected and stored at -70°C. According to the instructions of the kit, enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of cytokines IL-4 (Xinbosheng, catalog number: EMC003), IL-6 (Xinbosheng, catalog number: EMC004), IFN-γ (Xinbosheng, catalog number: EMC101g) and TNF-α (Xinbosheng, catalog number: EMC102a) in the spleen cell culture supernatant. The results are as follows Figure 7 As shown, FliC S.T -The levels of TNF-α, IFN-γ, IL-4 and IL-6 in the spleen cells of immune mice induced by the TGEV-S fusion protein immunization group were significantly higher than those in the PBS control group, indicating that the fusion protein immunization can effectively stimulate the immune mice to produce high levels of cytokines and induce a good cellular immune response.

[0052] (4) Neutralizing antibody detection After the serum to be tested was inactivated in a 56°C water bath for 30 minutes, it was serially diluted 2-fold (1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256) with MEM medium. Subsequently, the TGEV virus solution (SHXB strain) was diluted to 100 TCID 50 / 0.1 mL. The diluted virus solution and serum were mixed in a 1:1 ratio and placed in a 37°C incubator for reaction for 2 hours. The serum and virus solution mixture after neutralization reaction was inoculated into a 96-well ST cell culture plate that had formed a good monolayer. 4 wells were set up for each dilution and 100 μL of the mixture was added. A normal cell control group and a virus control group were set up for comparative experiments. After incubating the culture plate in a 37°C incubator for 1.5 hours, the liquid was discarded, and 100 μL of maintenance solution was added to each well and cultured for another 5 days. The cytopathic effect (CPE) was observed and recorded daily. Finally, the Reed-Muench method was used to calculate the serum neutralizing antibody titer. The results are as follows Figure 8 As shown, FliC S.T -The neutralizing antibody titer of the TGEV-S fusion protein immunization group was 1:2 6 , indicating that the fusion protein has good immunogenicity and can be used as a potential candidate epitope vaccine for TGEV.

[0053] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A FliC S.T -TGEV-S fusion protein, characterized in that The FliC S.T The amino acid sequence of the -TGEV-S fusion protein is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.

4.

2. The FliC according to claim 1 S.T -TGEV-S fusion protein, characterized in that The FliC S.T -TGEV-S fusion protein has an amino acid sequence as shown in SEQ ID NO.5 and a nucleotide sequence as shown in SEQ ID NO.

6. S.T As a skeleton protein; replace FliC with the dominant B cell antigen epitope in the RBD region of the TGEV S protein with the amino acid sequence as shown in SEQ ID NO.1 and the nucleotide sequence as shown in SEQ ID NO.2 S.T Construction of B cell antigen epitopes with low immunogenicity and exposed on the protein surface.

3. FliC according to any one of claims 1 to 2 S.T - A method for constructing a TGEV-S fusion protein, characterized in that: The specific steps include: (1) Synthetic plasmid pUC57-FliC S.T -TGEV-S was used as the amplification template for PCR amplification to obtain F S.T -TGEV- S Target gene fragment; (2) Combine pET-28α(+) plasmid and purified F S.T -TGEV-S The target gene fragment was subjected to double restriction digestion reaction, and then the purified target gene fragment was mixed with pET-28α(+) plasmid product and T4 DNA ligase to obtain a ligation product, which was then transformed into DH5α competent cells for culture to obtain the recombinant plasmid pET-28α-FliC S.T -TGEV-S; (3) The recombinant expression plasmid pET28α-FliC S.T -TGEV-S was transferred into Escherichia coli for culture, induced for expression, and then ultrasonically broken and centrifuged for purification to obtain the FliC S.T -TGEV-S fusion protein.

4. The construction method according to claim 3, characterized in that: In step (1), the primer sequences used in the PCR amplification process are shown in SEQ ID NO.7-8.

5. The construction method according to claim 3, characterized in that: In step (2), the double enzyme digestion reaction system is configured as follows: 60 μL of the target gene fragment or the plasmid, 2 μL each of BamHI and SacI, 10 μL of 10×CutSmart Buffer, and 26 μL of ddH2O. The above reagents are added to a centrifuge tube in sequence, mixed thoroughly, and reacted in a 37°C constant temperature water bath for 3 hours.

6. A porcine transmissible gastroenteritis virus epitope vaccine, characterized in that: The porcine transmissible gastroenteritis virus epitope vaccine comprises the FliC S.T -TGEV-S fusion protein.

7. The FliC according to any one of claims 1 to 2 S.T -Application of TGEV-S fusion protein in the preparation of drugs for preventing and treating porcine transmissible gastroenteritis.

8. The use according to claim 7, characterized in that The medicament includes a vaccine or an antibody.

9. The use according to claim 7, characterized in that The FliC S.T -TGEV-S fusion protein can induce the production of neutralizing antibodies against TGEV after being used to immunize mice.

10. The use according to claim 7, characterized in that The FliC S.T -TGEV-S fusion protein can be detected by anti-FliC S.T and recognized by polyclonal antibodies against TGEV S protein.

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