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

Abstract

The present application relates to the field of biotechnology, and particularly relates to a porcine transmissible gastroenteritis virus epitope vaccine, a preparation method and application thereof, and FliC S.T The amino acid sequence of the TGEV-S fusion protein is shown as SEQ ID NO. 3, and the nucleotide sequence is shown as SEQ ID NO. 4. The fusion protein FliCS.T-TGEV-S prepared in the present application is an epitope vaccine based on the dominant antigen epitope of TGEV, which combines the immunodominant epitope, fusion protein technology and advanced genetic engineering methods to enhance the specificity and protection effect of the vaccine. The vaccine can not only induce high-level antibody response and effectively inhibit TGEV infection, but also has high safety and stability. Compared with the traditional vaccine, the vaccine of the present application has significant advantages in production cost, immunization efficacy, protection durability and the like, and provides a feasible TGE prevention and control scheme for the pig industry.

Description

TECHNICAL FIELD

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

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

[0003] TGEV belongs to the Coronaviridae family and is a single-stranded positive-sense RNA virus with an envelope, 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, especially its receptor binding domain (RBD), which can bind to the receptors of pig intestinal epithelial cells, becoming a key to viral invasion. Studies have shown that by blocking the binding of the S protein to host cells, viral infection can be effectively inhibited. Therefore, the RBD of the S protein is considered an ideal target for TGEV vaccine development. TGEV rapidly replicates in intestinal epithelial cells after infection, causing acute enteritis and mucosal damage. Because the immune system of piglets is not yet mature, diarrhea, dehydration, and electrolyte imbalance caused by TGEV infection are particularly deadly in piglets. In addition, TGEV infection can reduce the growth efficiency and feed conversion rate of pig populations, increasing the burden on breeding.

[0004] Current vaccines for TGEV prevention and control mainly include inactivated vaccines, attenuated vaccines, and genetically engineered subunit vaccines: Inactivated vaccines are prepared by killing the whole virus of TGEV through chemical or physical methods. Although relatively safe, the induced immune response is weak, the immune protection effect is limited, and multiple vaccinations are required to maintain long-term protection. Attenuated vaccines are prepared from attenuated TGEV and can replicate in the body to a limited extent, inducing a relatively persistent immune response. However, due to the residual virulence of attenuated vaccines, there is a potential safety hazard, especially in piglets with weak immunity, which may cause mild infection. In addition, the production cycle of attenuated vaccines is long, and the quality control is difficult. Genetically engineered subunit vaccines are constructed by expressing key antigen proteins or antigen epitopes of the virus, which are relatively safe and can induce specific immune responses against TGEV through precise selection of antigen epitopes. However, current vaccines still have limitations in terms of immunization 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 effect, poor immune persistence, and high cost. These limitations have prompted researchers to continue exploring new, efficient, and safe TGEV vaccines.

[0005] Epitope vaccines are a new vaccine technology that mainly targets the most immunogenic antigen epitopes in pathogens to induce highly specific immune responses. This vaccine development strategy is expected to overcome the limitations of traditional vaccines and significantly improve vaccine safety and immunization efficacy. The advantages of epitope vaccines mainly include the following points: (1) strong targeting: Epitope vaccines can precisely activate the host immune system by selecting viral antigen epitopes, avoiding non-specific reactions caused by non-key antigens. This makes the design of epitope vaccines more targeted and reduces the risk of adverse reactions. (2) Enhanced immunogenicity: Epitope vaccines use immunodominant B-cell epitopes in TGEV S protein to induce high levels of neutralizing antibodies, blocking the receptor binding process of the virus and effectively inhibiting viral infection. Compared with traditional vaccines, epitope vaccines have more significant immunization 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, especially suitable for large-scale application. Epitope vaccine technology has shown good application prospects in multiple fields and has been proven to be superior in controlling viral infections in research. 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

[0006] The present application aims at providing a porcine transmissible gastroenteritis virus epitope vaccine, a preparation method and application thereof, which is derived from a dominant B cell antigen epitope in the RBD region of TGEV S protein, solves the problem of unsatisfactory effect of the existing vaccine, and greatly improves the immunization effect and safety of the vaccine through unique fusion protein design and autologous adjuvant effect.

[0007] The present application aims at providing a porcine transmissible gastroenteritis virus epitope vaccine, a preparation method and application thereof, which is derived from a dominant B cell antigen epitope in the RBD region of TGEV S protein, solves the problem of unsatisfactory effect of the existing vaccine, and greatly improves the immunization effect and safety of the vaccine through unique fusion protein design and autologous adjuvant effect.

[0008] The present application 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 as SEQ ID NO. 3, and the nucleotide sequence is shown as SEQ ID NO. 4.

[0009] Further, the FliC S.T The TGEV-S fusion protein is FliC S.T The dominant B cell antigen epitope in the RBD region of the TGEV S protein is replaced by the FliC S.T which has low immunogenicity and is exposed to the surface of the protein.

[0010] The present application also provides a construction method of the FliC S.T -TGEV-S fusion protein, which specifically comprises the following steps:

[0011] (1) taking the synthetic plasmid pUC57-FliC S.T -TGEV-S as an amplification template to perform PCR amplification, so as to obtain FliC S.T - TGEV-S the target gene fragment;

[0012] (2) performing double enzyme digestion reaction on the pET-28α(+) plasmid and the purified FliC S.T -TGEV-S target gene fragment, then mixing the purified target gene fragment, the pET-28α(+) plasmid product and T4 DNA ligase to obtain a ligation product, and then culturing in DH5α competent cells to obtain a recombinant plasmid pET-28α-FliC S.T -TGEV-S;

[0013] (3) the recombinant expression plasmid pET28a-FliC S.T -TGEV-S is introduced into E. coli for culture, expression induction, ultrasonic crushing, centrifugal separation and purification, and the FliC is obtained. S.T -TGEV-S fusion protein.

[0014] Further, in step (1), the primer sequence used in the PCR amplification process is shown in SEQ ID NO. 7-8.

[0015] Further, in step (2), the double enzyme digestion reaction system is configured as: the target gene fragment or the plasmid 60 μL, BamHI and SacI each 2 μL, 10×CutSmart Buffer 10 μL, ddH2O 26 μL, the above reagents are sequentially added into a centrifugal tube, fully mixed, and then reacted in a 37℃ constant temperature water bath for 3 hours.

[0016] The application also provides a porcine transmissible gastroenteritis virus epitope vaccine, which comprises the FliC S.T -TGEV-S fusion protein.

[0017] The application also provides the FliC S.T The application also provides application of the FliC

[0018] Further, the medicine comprises a vaccine or an antibody.

[0019] Further, the FliC S.T After the FliC

[0020] Further, the FliC S.T The TGEV-S fusion protein can be recognized by anti-FliC S.T and anti-TGEV S protein polyclonal antibodies.

[0021] The FliC S.T The TGEV-S fusion protein has the following beneficial effects:

[0022] (1) Autologous adjuvant effect: using Salmonella typhimurium flagellin (FliC) with adjuvant effect as the skeleton, the functional fusion expression of the dominant B cell epitope of the RBD region of TGEV S protein and FliC is realized for the first time. FliC, as an immune stimulating molecule, can significantly activate the innate immune and adaptive immune response of the host, so that the fusion protein vaccine can induce the production of neutralizing antibodies while stimulating the response of cellular immunity. By adding the FliC skeleton to the fusion protein vaccine, the immune protection effect of the vaccine on TGEV can be significantly enhanced. The fusion protein can achieve significant immune effect without additional adjuvant, thereby reducing the production cost and simplifying the immune operation.

[0023] (2) Enhanced immune protection effect: the fusion protein of the present application can not only induce efficient humoral immunity, but also stimulate cellular immunity, thereby improving the protection effect of the vaccine. The fusion protein can effectively induce neutralizing antibodies against TGEV and enhance the cellular immune response, thereby providing comprehensive immune protection for piglet populations.

[0024] (3) Excellent production characteristics: the production process of the fusion protein is simple and low in cost, and is suitable for large-scale popularization and application. Compared with the high cost and complex process of traditional vaccines, the vaccine of the present application has good production efficiency, and significantly saves manpower and resources.

[0025] In summary, the fusion protein FliCS.T-TGEV-S prepared in the present application is an epitope vaccine based on the dominant antigen epitope of TGEV, which combines the immune dominant epitope, fusion protein technology and genetic engineering and other advanced means to enhance the specificity and protection effect of the vaccine. The vaccine can not only induce high-level antibody response and effectively inhibit TGEV infection, but also has high safety and stability. Compared with traditional vaccines, the vaccine of the present application has significant advantages in production cost, immune efficacy, protection durability and the like, and provides a feasible TGE prevention and control scheme for the pig industry. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0027] Figure 1 is a schematic diagram for constructing F18-PEDV-S chimeric pilus, wherein E represents the dominant neutralizing B cell antigen epitope of the RBD region of PEDV S protein.

[0028] Figure 2Figure 1 is a result of PCR amplification of FliCS.T-TGEV-S chimeric gene, wherein lane M is 2K Plus II DNA Marker; lane 1 is the PCR amplification result of FliCS.T-TGEV-S chimeric gene.

[0029] Figure 3 Figure 2 is an electrophoretogram of PCR identification of FliCS.T-TGEV-S recombinant strain, wherein lane M is 2K Plus II DNA Marker; lanes 1-4 are the PCR amplification results of different clones of FliCS.T-TGEV-S recombinant strain.

[0030] Figure 4 Figure 3 is a SDS-PAGE map of purified FliCS.T-TGEV-S fusion protein, FliCS.T and TGEV-S protein, wherein M is protein molecular weight standard; lane 1 is purified FliCS.T-TGEV-S fusion protein; lane 2 is purified FliCS.T recombinant protein; lane 3 is purified TGEV-S recombinant protein.

[0031] Figure 5 Figure 4 is a Western blot map of purified FliCS.T-TGEV-S fusion protein, FliCS.T and TGEV-S protein.

[0032] Figure 6 Figure 5 is a result of ELISA detection of specific anti-TGEV-S IgG antibody level in serum of FliCS.T-TGEV-S fusion protein immunized mice, wherein * indicates P<0.05 and ** indicates P<0.01.

[0033] Figure 7 Figure 6 is a result of ELISA detection of expression level of inflammatory cytokines IL-4, IL-6, IFN-γ and TNF-α in spleen cells of immunized mice.

[0034] Figure 8 Figure 7 is a result of ELISA detection of neutralizing antibody titer of TGEV (SHXB) virus in serum of immunized mice. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present application will now be described in detail with reference to the drawings. The detailed description is not to be considered to limit the application in any way, but rather to explain certain aspects, features and embodiments of the application.

[0036] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, concentrations, solvent amounts, and the like, there are intended to be included in the present application each and every intermediate value of the range. For example, a range of 1 to 2 includes each and every intermediate value and each "sub-range" between (and including) the upper and lower limits of the range. The upper and lower limits of these sub-ranges can independently be included or excluded in the range, and are also endpoints of the range, so that the stated range is a continuum between the stated limits. Other objects, embodiments, and advantages of the present application are set forth in part in the description that follows, and in part will be obvious from the description, or can be learned by practice of the application.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in the specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application.

[0038] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only. It is intended to output the full scope of the application solely based on the claims.

[0039] With respect to the terms "comprising", "containing", "having", "including", and the like used herein, these terms are used in their open-ended, non-limiting sense to encompass the presence of one or more elements, features, or components, but do not preclude the presence or addition of one or more other elements, features, or components.

[0040] Chemicals, biochemicals and materials used in the present application are commercially available unless otherwise specified.

[0041] Example 1 FliC S.T Construction, expression and identification of TGEV-S fusion gene

[0042] (1) FliC S.T Construction and structure prediction of TGEV-S fusion protein

[0043] The RBD region of TGEV S protein was predicted for B cell epitopes by using the bioinformatics online tool IEDB (http: / / www.iedb.org / ). FliC S.T The construction of TGEV-S fusion protein is shown in the schematic diagram as Figure 1 Specifically, FliC S.T(bacterial flagellin protein) (the amino acid sequence of which is shown in SEQ ID NO. 5, and the gene sequence of which is shown in SEQ ID NO. 6) as a scaffold protein, and the dominant B cell epitope in the RBD region of TGEV S protein (the amino acid sequence of which is shown in SEQ ID NO. 1, and the gene sequence of which is shown in SEQ ID NO. 2) is replaced by FliC S.T The B cell epitope with low immunogenicity and exposed on the surface of the protein is replaced by FliC S.T The TGEV-S fusion protein (the amino acid sequence of which is shown in SEQ ID NO. 3, and the gene sequence of which is shown in SEQ ID NO. 4) is obtained.

[0044] Subsequently, the structure of the fusion protein is modeled by using the Phyre2 portal, and the model is scored by using key parameters such as reliability, linear coverage, and homology (>90%), so as to screen the FliC S.T The TGEV-S protein model is used as the final model. The results show that the inserted exogenous B cell epitope does not destroy the natural conformation of the scaffold protein FliC S.T , and the epitope is located on the surface of FliC S.T The TGEV-S fusion protein, which helps to achieve the expected antigen presentation effect.

[0045] (2) FliC S.T -TGEV-S PCR amplification of the chimeric gene

[0046] The synthetic plasmid pUC57-FliC S.T The TGEV-S is used as an amplification template, and primers P1 (SEQ ID NO. 7) and primers P2 (SEQ ID NO. 8) are used for PCR amplification, and the FliC S.T -TGEV-S chimeric gene fragment is successfully obtained.

[0047] 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, 2.5 mM dNTP mixture 4 μL, ddH2O 27 μL, and 2 μL of each of the upstream and downstream primers, with a total reaction volume of 50 μL.

[0048] PCR reaction conditions were as follows: initial denaturation at 95°C for 5 minutes, followed by 31 cycles of denaturation at 94°C for 30 seconds, annealing at 54°C for 30 seconds, extension at 72°C for 2 minutes, and a final extension at 72°C for 7 minutes, followed by storage at 4°C. Notably, primers P1 and P2 contain restriction sites for BamHI and SacI, respectively, to facilitate subsequent cloning operations.

[0049] PCR amplification results are shown in Figure 2 ,Depend on Figure 2 The target band was observed at 1536 bp, consistent with the expected result. The target fragment was purified and recovered using the Tiangen Biochemical Technology Co., Ltd. DNA Gel Recovery Kit (Cat. No. DP214-03), providing a high-quality DNA template for subsequent experiments.

[0050] (3) pET-28α-FliC S.T Construction of TGEV-S recombinant plasmid

[0051] First, the pET-28α(+) plasmid and the purified FliC S.T -TGEV-S The target gene fragment was double-digested with BamHI (NEB, Cat. No. R3136V) and SacI (NEB, Cat. No. R3156V). The digestion reaction system was as follows: 60 μL of gene fragment or plasmid, 2 μL each of BamHI and SacI, 10 μL of 10× CutSmart Buffer, and 26 μL of ddH2O. These reagents were added sequentially to a centrifuge tube, mixed thoroughly, and incubated in a 37°C water bath for 3 hours.

[0052] 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.

[0053] The ligation reaction system is as follows: 3 μL of pET-28α(+) plasmid digestion product, FliC 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.

[0054] The ligation product was then transformed into DH5a competent cells, 800 μL of LB liquid medium without resistance was added, and the cells were cultured at 37°C and 220 rpm for 1 h. An appropriate amount of bacterial solution was taken and spread on LB solid medium containing kanamycin sulfate, and cultured at 37°C for 16 h. 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.

[0055] The bacterial solution was used as a template for preliminary identification by PCR. As shown in Figure 3 , the four clones picked all showed a target band at about 1536 bp, which was consistent with the expectation. After confirming the positive clones by PCR identification, the samples were sent to a company for DNA sequencing. The final sequencing result verified the accuracy of the clone construction, and the successfully constructed recombinant plasmid was named pET-28a-FliC S.T -TGEV-S.

[0056] (4) Expression and identification of FliC S.T -TGEV-S fusion protein

[0057] The recombinant expression plasmid pET28a-FliC S.T -TGEV-S was transformed into E. coli BL21 (DE3), and a single colony was inoculated into 5 mL of LB medium containing kanamycin sulfate and cultured at 37°C and 220 rpm for 12 h. Then, primers P1 (SEQ ID NO. 7) and P2 (SEQ ID NO. 8) were used for PCR identification, and the correct clone was obtained after confirming the correct clone, i.e., the 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 at 37°C with shaking until the OD600 value reached about 0.6. Then, IPTG was added at a final concentration of 1 mmol / L, and expression was induced for 4 h. The induced bacterial cells were collected, broken by ultrasonic wave, and the precipitate was collected after centrifugation. The inclusion body lysis solution was used for overnight lysis. After centrifugation again, the supernatant was collected and the protein was purified by nickel ion affinity chromatography. The purified sample was detected by SDS-PAGE electrophoresis, and the results are shown in Figure 4 , which showed a clear band at about 54 kDa. After determining the protein concentration, the purified FliC S.T -TGEV-SS fusion protein was stored at -70°C for standby.

[0058] For further analysis, the purified FliC S.T- TGEV-S fusion protein sample was loaded on 12% separation gel for SDS-PAGE, and after the electrophoresis was completed, the protein was transferred to a PVDF membrane. The membrane was blocked in a 5% skim milk solution at room temperature for 1 hour, and then incubated with 1:3000 diluted anti-TGEV S protein polyclonal antibody and 1:6000 diluted anti-Salmonella typhimurium flagellin polyclonal antibody (primary antibody, both self-made in the laboratory) respectively at 4°C overnight. The next day, 5 washes were performed with PBST for 8 minutes each time, then 1:5000 diluted goat anti-mouse IgG-HRP secondary antibody (ABclonal, item number: AS003) was added and incubated at 37°C for 90 minutes, 5 washes were performed again with PBST for 8 minutes each time, and finally color development was performed with an ECL color development kit. As shown in Figure 5 FliC S.T - The TGEV-S fusion protein can be recognized by anti-TGEV S protein polyclonal antibody and anti-Salmonella typhimurium flagellin polyclonal antibody, indicating that the expression of the fusion protein retains the biological activity of the B cell epitopes of FliC S.T and TGEV S protein.

[0059] Example 2 FliC S.T Immunogenicity analysis of TGEV-S fusion protein

[0060] (1) Mouse immunization

[0061] Fifteen 6-week-old BALB / c male mice were randomly divided into 3 groups, 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 was subcutaneously immunized with the recombinantly expressed TGEV S protein mixed and emulsified with an equal volume of Freund's complete adjuvant, and the immunization dose was 50 μg per mouse; the third group was used as a control group and was subcutaneously injected with 100 μL of sterilized 0.01 M PBS buffer (pH=7.4). All mice were immunized a total of three times. After the first immunization, a booster immunization was performed every two weeks, and the third group used incomplete Freund's adjuvant at the second and third immunizations. Before immunization (0 days) and on the 7th day, 14th day, 21st day, 28th day, 35th day, and 42nd day after the first immunization, blood was collected from the retro-orbital vein, and the serum was stored at -20°C. Fourteen days after the last immunization (i.e. the third immunization), the mice were euthanized by cervical dislocation, and the spleen cells were isolated for detection of inflammatory cytokines.

[0062] (2) Anti-TGEV-S specific IgG antibody detection

[0063] The recombinant TGEV S protein expressed in our laboratory was used as the coating antigen, and was coated on a 96-well enzyme-labeled 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 with PBST for 3 times, and then was blocked with 10% skim milk solution at 37°C for 1 hour. After the blocking solution was discarded, the plate was washed with PBST for 3 times, and then the immune serum was added for 2-fold serial dilution (1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800), 100 μL of which was added to each well, and incubation was performed at 37°C for 90 minutes. The plate was washed with PBST for 3 times, and then goat anti-mouse IgG-HRP (secondary antibody, ABclonal, item number: AS003) diluted at 1:5000 was added to each well, 100 μL of which was added, and incubation was performed at 37°C for 90 minutes. After washing for 3 times, TMB color developing reagent was added, and color development was performed at 37°C for 30 minutes in the dark, and the OD650nm value of each well was immediately read by an enzyme-labeled instrument. The results are shown in Figure 6 Figure 6. The FliC S.T The TGEV-S fusion protein immune group and the TGEV S protein and Freund's adjuvant immune group could induce high levels of anti-TGEV S protein specific IgG antibody production from the 7th day, while the PBS control group could not detect the corresponding antibody. This result shows that the FliC S.T The TGEV-S fusion protein has good immunogenicity.

[0064] (3) Detection of cytokines in the spleen cell culture supernatant

[0065] Fourteen days after the last immunization, the mice were sacrificed by the painless cervical dislocation method, and then the spleen was aseptically removed and placed in a culture dish containing PBS. The spleen was ground using a syringe piston, and filtered through a 200-mesh sieve to collect the cell suspension. The suspension was centrifuged at 1500 rpm for 10 minutes, and the supernatant was discarded. After removing red blood cells using red blood cell lysis solution, 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 Then, 200 μL of the cell suspension was inoculated into a 6-well cell culture plate, 5 μg / mL of the corresponding protein was added to each well to stimulate the spleen cells, and incubation was performed at 37°C in a 5% CO2 incubator for 72 hours. After the incubation, the supernatant was collected and stored at -70°C. According to the kit instructions, the concentrations of cytokines IL-4 (Xinboseng, item number: EMC003), IL-6 (Xinboseng, item number: EMC004), IFN-γ (Xinboseng, item number: EMC101g), and TNF-α (Xinboseng, item number: EMC102a) in the spleen cell culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA). The results are shown inFigure 7 FliC S.T The contents of TNF-α, IFN-γ, IL-4 and IL-6 in the spleen cells of the immune mice induced by the TGEV-S fusion protein immune group were significantly higher than those of the PBS control group, indicating that the fusion protein after immunization can effectively stimulate the immune mice to produce high levels of cytokines and induce good cellular immune response.

[0066] (4) Neutralizing antibody detection

[0067] After inactivating the serum to be tested in a 56℃ water bath for 30 minutes, the serum was diluted by 2 times in series (1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256) with MEM medium. Then, the TGEV virus liquid (SHXB strain) was diluted to 100 TCID 50 / 0.1 mL with MEM medium. The diluted virus liquid and the serum were mixed in a ratio of 1:1, and were placed in a 37℃ incubator for reaction for 2 hours. The serum and virus liquid mixture after neutralization reaction was inoculated into a 96-well ST cell culture plate with a good monolayer, 4 holes were set for each dilution, and 100 μL of the mixture was added. Normal cell control group and virus control group were set up for comparative experiments. After placing the culture plate in a 37℃ incubator for 1.5 hours, the liquid was discarded, and 100 μL of maintenance liquid was added to each hole, and the culture was continued for 5 days. The cytopathic effect (CPE) was observed and recorded every day. Finally, the serum neutralizing antibody titer was calculated by the Reed-Muench method. The results are shown in Table 2. Figure 8 FliC S.T The neutralizing antibody titer of the TGEV-S fusion protein immune group was 1:2 6 , indicating that the fusion protein has good immunogenicity and can be used as a potential candidate epitope vaccine of TGEV.

[0068] The above-described embodiments only express several embodiments of the present application, which are described in detail and specifically, but cannot be understood as limiting the scope of the patent. It should be noted that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which are all within the protection scope of the present application. Therefore, the protection scope of the present patent should be subject to the appended 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. FliC as claimed in claim 1 S.T -The method for constructing TGEV-S fusion protein is characterized in that, The specific steps include: (1) Synthesize plasmid pUC57-FliC S.T -TGEV-S was used as the amplification template for PCR amplification to obtain FliC S.T -TGEV- S Target gene fragment; (2) Combine the pET-28α(+) plasmid and the purified FliC S.T -TGEV-S The target gene fragment was subjected to double enzyme digestion reaction, and 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 and cultured 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 and induced expression, and then ultrasonically broken and centrifuged for purification to obtain the FliC S.T -TGEV-S fusion protein.

3. The construction method according to claim 2, wherein: In step (1), the primer sequences used in the PCR amplification process are shown in SEQ ID NO. 7-8.

4. The construction method according to claim 2, wherein: 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.

5. 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.

6. The FliC according to claim 1 S.T -Application of TGEV-S fusion protein in the preparation of drugs for preventing and treating porcine transmissible gastroenteritis.

7. The use according to claim 6, characterized in that The drugs include vaccines or serum neutralizing antibodies.

8. The use according to claim 6, characterized in that The FliC S.T -TGEV-S fusion protein can induce the production of serum neutralizing antibodies against TGEV after being used to immunize mice.

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