Porcine rotavirus VP8 protein dominant B cell neutralizing epitope peptide, multi-epitope fusion protein and application thereof

Abstract

The invention discloses a porcine rotavirus VP8 protein dominant B cell neutralizing epitope peptide, a multi-epitope fusion protein and application of the multi-epitope fusion protein. The FliCS.T-VP8 multi-epitope fusion protein prepared by the invention is a multi-epitope fusion protein which takes salmonella typhimurium flagellin FliCS.T as a molecular skeleton and integrates the dominant B cell neutralizing epitope of the VP8 protein of the porcine rotavirus. According to the design, immunodominance epitope screening, multi-epitope fusion protein construction and genetic engineering technologies are fused, and the immunogenicity and immune protection efficacy of the fusion protein are remarkably enhanced. The FliCS.T-VP8 multi-epitope fusion protein not only can effectively induce high-level VP8 protein specific antibody response and cellular immune response, but also shows good porcine rotavirus neutralizing activity. In addition, the fusion protein has relatively high safety and stability, and has the potential of being used as an efficient subunit vaccine for preventing and controlling porcine rotavirus infection in the pig industry.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a porcine rotavirus VP8 protein dominant B cell neutralizing epitope peptide, a multi-epitope fusion protein and applications thereof. Background Art

[0002] Porcine rotavirus (PoRV) infection causes acute gastroenteritis in piglets, a major disease that affects piglet production performance and economic benefits. Four serogroups have been identified: RVA, RVB, RVC, and RVH. RVA accounts for over 90% of clinical detections and remains the dominant serogroup. Recent studies have shown that the G9 serotype has replaced the traditional G5 serotype as the dominant prevalent type, accounting for over 80% of cases. The P serotype is dominated by P

[13] and P

[23] , which together account for 82.1%. The coexistence of multiple serotypes makes it difficult for vaccines developed based on a single serogroup to achieve full coverage, significantly increasing the complexity and difficulty of immunization prevention and control.

[0003] PoRV possesses a high degree of genetic diversity, rooted in its unique mechanism of segmental rearrangement. As a segmented, double-stranded RNA virus, the PoRV genome consists of 11 RNA segments, approximately 18.5 kb in length, encoding six structural proteins (VP1-VP4, VP6, and VP7) and six nonstructural proteins (NSP1-NSP6). This segmented structure not only enables the virus to generate novel gene combinations through gene rearrangement, but also allows point mutations to accumulate during gene replication, continuously generating new variants. PoRV poses a particularly serious health risk to piglets. The virus specifically infects mature epithelial cells of the small intestinal villi. Its invasion is mediated by the VP4 protein: after trypsin treatment, VP4 is cleaved into VP5 and VP8, which work synergistically in viral attachment and membrane penetration. The VP8 subunit recognizes and binds to receptors by binding to sialic acid residues or tissue-blood group antigens (HBGAs) on the cell surface. After the virus enters cells, it triggers villus atrophy and crypt hyperplasia, leading to impaired nutrient absorption and imbalanced intestinal osmotic pressure. Given that the VP8 subunit is rich in major neutralizing epitopes, it has become a core target for PoRV vaccine design and antibody development.

[0004] Currently, comprehensive prevention and control strategies for PoRV infection primarily encompass symptomatic drug treatment, biosafety management, and vaccination. Since there are no specific antiviral drugs for PoRV, symptomatic supportive care, such as oral rehydration salts, tannic acid protein, and broad-spectrum antibiotics, is commonly used clinically to alleviate dehydration and secondary infection. Biosafety measures focus on isolating sick pigs, strictly controlling the flow of personnel and vehicles, regularly disinfecting farms, and implementing scientific animal husbandry management to minimize transmission. However, given the high genetic diversity and frequent gene rearrangement of PoRV, these passive prevention and control measures often only temporarily alleviate clinical symptoms and are unable to address the challenges posed by increased virulence and antigenic drift. In contrast, vaccination is considered a core solution for preventing PoRV infection due to its cost-effectiveness, ability to achieve a herd immunity barrier, and long-lasting protection. Currently available vaccines primarily include inactivated vaccines, live attenuated vaccines, and novel genetically engineered vaccines. However, due to the multiple serotypes of PoRV and the limited cross-protection between different genotypes, coupled with the virus's high variability, interspecies transmission, and frequent rearrangement, the breadth and durability of protection provided by existing vaccines remain insufficient for practical application. Therefore, the development of a new generation of PoRV vaccines that can cover major serotypes and possess both safety and broad-spectrum efficacy is imperative. In recent years, bacterial flagellin has garnered widespread attention due to its excellent immune adjuvant properties. Not only can this protein simultaneously stimulate humoral and cellular immunity, overcome oral tolerance, and significantly promote the secretion of mucosal proinflammatory cytokines, but its high structural plasticity also allows for the insertion of exogenous epitopes in multiple functional domains without disrupting the native conformation, thereby achieving a multi-pathway synergistically enhanced adjuvant effect.

[0005] Although flagellin has shown significant advantages as an adjuvant, it still faces two core challenges in its actual application. The first is that flagellin has a high immunogenicity, which may lead to excessive immune and inflammatory responses, thereby causing potential damage to the body. In addition, pre-existing antibodies against flagellin may induce immune tolerance through neutralization, further leading to a weakening of the vaccine effect. Therefore, how to balance the immunogenicity and safety of flagellin is an important difficulty in its practical application. Secondly, although truncating the hypervariable region of flagellin or inserting exogenous genes can reduce its immunogenicity, this genetic modification may destroy the structure of the TLR5 binding domain, thereby weakening its adjuvant efficacy. Therefore, when performing genetic modification, how to effectively maintain the structural stability and functional activity of flagellin remains an important technical challenge faced in the development process. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a dominant B cell antigen epitope peptide of porcine rotavirus VP8 protein or a combination thereof.

[0007] The technical problem that the present invention also aims to solve is to provide a nucleic acid molecule encoding the B cell antigen epitope peptide or a combination thereof.

[0008] The technical problem that the present invention also solves is to provide a FliC S.T -VP8 multi-epitope fusion protein and preparation method thereof.

[0009] The technical problem that the present invention also solves is to provide a FliC encoding S.T -Nucleic acid molecule of VP8 multi-epitope fusion protein.

[0010] The technical problem to be solved by the present invention is to provide a dominant B cell antigen epitope peptide or a combination thereof, a nucleic acid molecule of the B cell antigen epitope peptide or a combination thereof, the FliC S.T -VP8 multi-epitope fusion protein, encoding the FliC S.T -Application of nucleic acid molecules of VP8 multi-epitope fusion protein in the preparation of drugs for preventing or treating porcine rotavirus infection.

[0011] The technical problem that the present invention also aims to solve is to provide a vaccine or a neutralizing antibody or a diagnostic reagent.

[0012] Technical solution: The present invention provides a dominant B cell antigen epitope peptide of porcine rotavirus VP8 protein or a combination thereof, wherein the amino acid sequence of the B cell antigen epitope peptide or the combination thereof comprises at least one of SEQ ID NO.1 to SEQ ID NO.4.

[0013] The nucleic acid molecule encoding the B cell antigen epitope peptide or a combination thereof, wherein the DNA sequence of the nucleic acid molecule encoding the B cell antigen epitope peptide or a combination thereof comprises at least one of SEQ ID NOs. 5 to 8.

[0014] The present invention also includes a FliC S.T -VP8 multi-epitope fusion protein, the FliC S.T -VP8 multi-epitope fusion protein with Salmonella typhimurium flagellin FliC S.T As a skeleton protein, the dominant B cell antigen epitope peptide or its combination is substituted for FliC S.T The B cell antigen epitope with low immunogenicity and exposed on the protein surface is constructed. Preferably, the FliC S.T -VP8 multi-epitope fusion protein is a protein that combines the dominant B cell antigen epitope peptide in series with the Salmonella typhimurium flagellin FliC S.T In terms of the backbone, preferably, the amino acid sequence of the multi-epitope fusion protein is as shown in SEQ ID NO.9.

[0015] The present invention also includes encoding the FliCS.T -A nucleic acid molecule of a VP8 multi-epitope fusion protein, the DNA sequence of which is shown in SEQ ID NO.10.

[0016] The present invention also includes the FliC S.T -The preparation method of VP8 multi-epitope fusion protein comprises the following steps:

[0017] (1) Obtain FliC S.T -VP8 target gene fragment;

[0018] (2) The FliC S.T -VP8 target gene fragment is connected with plasmid to obtain recombinant plasmid;

[0019] (3) The recombinant plasmid is transferred into Escherichia coli for culture and induction of expression, and then the multi-epitope fusion protein is obtained after ultrasonic disruption and centrifugal separation and purification.

[0020] Wherein, the acquisition of nucleic acid molecules in step (1) includes: synthesizing plasmid pUC57-FliC S.T -VP8 was used as a template for PCR amplification to obtain FliC S.T -VP8 target gene fragment, the primer sequences used in the PCR amplification are shown in SEQ ID NO.13 and SEQ ID NO.14.

[0021] Wherein, step (2) is as follows: pET28a(+) plasmid and purified FliC obtained in step (1) are respectively S.T -VP8 target gene fragment was double-digested; the purified target gene fragment was mixed with the pET28a(+) plasmid vector after enzyme digestion, and T4 DNA ligase was added to carry out ligation reaction to obtain a ligation product; the ligation product was transformed into TOP10 competent cells, and the recombinant plasmid pET28a(+)-FliC was obtained after culture. S.T -VP8.

[0022] The present invention also includes the dominant B cell antigen epitope peptide or its combination, the nucleic acid molecule of the B cell antigen epitope peptide or its combination, the FliC S.T -VP8 multi-epitope fusion protein, encoding the FliC S.T -Application of nucleic acid molecules of VP8 multi-epitope fusion protein in the preparation of drugs for preventing or treating porcine rotavirus infection.

[0023] Wherein, the medicine includes a vaccine, an antibody or a diagnostic reagent.

[0024] The present invention also includes a vaccine or neutralizing antibody or diagnostic reagent, wherein the vaccine includes the FliCS.T -VP8 multi-epitope fusion protein; the neutralizing antibody comprises inducing the production of the multi-epitope fusion protein after immunizing an animal; the diagnostic reagent comprises FliC S.T -The immune serum of VP8 multi-epitope fusion protein specifically recognizes VP8 recombinant protein.

[0025] Beneficial effect: Compared with the prior art, the FliC S.T -VP8 multi-epitope fusion protein innovatively uses Salmonella typhimurium flagellin FliC S.T The invention is a molecular framework and endogenous adjuvant. By precisely embedding the dominant B cell neutralizing epitope of the VP8 protein of PoRV in its hypervariable region, the self-adjuvant effect of the fusion protein is utilized to significantly enhance immunogenicity. The invention has the following specific advantages:

[0026] (1) Self-adjuvant effect: The present invention is the first to use the Salmonella typhimurium flagellin FliC S.T Used as a backbone, fused with the dominant B cell neutralizing epitope of the VP8 protein of PoRV. S.T It possesses excellent immune adjuvant function, not only activating the host's innate immunity and accelerating the secretion of inflammatory factors, but also enhancing adaptive immunity, significantly increasing neutralizing antibody titers and T cell response levels. Because the fusion protein has its own adjuvant activity, it can achieve highly effective immunogenicity during vaccination without the need for traditional adjuvants, significantly reducing production costs and simplifying the vaccine preparation process.

[0027] (2) Enhanced immune protection effect: The multi-epitope fusion protein constructed by the present invention exhibits excellent performance at both humoral and cellular immune levels. On the one hand, the precise exposure of the VP8 neutralizing epitope can induce high-titer neutralizing antibodies against PoRV, effectively blocking viral infection; on the other hand, FliC S.T The skeleton promotes the secretion of Th1 / Th2 cytokines, stimulates a strong cellular immune response, and thus improves the body's comprehensive protection against PoRV.

[0028] In summary, the VP8 dominant B cell neutralizing epitope of PoRV was combined with FliC based on the MEFA technology platform. S.T The FliC constructed by the present invention is combined with the autologous adjuvant skeleton. S.T The VP8 multi-epitope fusion protein exhibits both highly effective neutralization and cellular immune activity. Compared to traditional inactivated or attenuated vaccines, this fusion protein vaccine offers significant advantages in production cost, immune efficacy, and ease of use, providing the swine industry with a broad-spectrum, long-lasting PoRV prevention strategy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 For FliC S.T-Schematic diagram of the construction of VP8 multi-epitope fusion protein and its predicted structure, where E1 to E4 represent the dominant B cell neutralizing epitopes of PoRV VP8 protein. A. FliC S.T -Schematic diagram of the construction of VP8 multi-epitope fusion protein, B.FliC S.T -VP8 multi-epitope fusion protein secondary structure prediction diagram; C.FliC S.T -Prediction of the tertiary structure of VP8 multi-epitope fusion protein.

[0030] Figure 2 For FliC S.T - PCR amplification results of VP8 chimeric gene, where lane M is 2K PlusⅡ DNA Marker; lane 1 is FliC S.T -VP8 chimeric gene; lane 2 is the negative control.

[0031] Figure 3 For the recombinant expression plasmid pET-28a(+)-FliC S.T -VP8 PCR identification electrophoresis diagram, where lane M is 2KPlusⅡDNAMarker; lane 1 is the recombinant expression plasmid pET-28a(+)-FliC S.T -PCR amplification results of VP8; lane 2 is the negative control.

[0032] Figure 4 For purified FliC S.T -SDS-PAGE of VP8 multi-epitope fusion protein, where M is the protein molecular weight standard; lane 1 is the purified FliC S.T -VP8 multi-epitope fusion protein; Lane 2 is purified FliC S.T Lane 3 is the purified VP8 recombinant protein.

[0033] Figure 5 This is a Western blot analysis of the purified VP8 recombinant protein. Figure 5 A is the reaction diagram of VP8 recombinant protein and anti-VP8 polyclonal antibody; Figure 5 B is VP8 recombinant protein and anti-FliC S.T - VP8 polyclonal antibody reactivity graph.

[0034] Figure 6 ELISA for detecting FliC S.T -Specific anti-VP8 antibody levels in the serum of mice immunized with VP8 multi-epitope fusion protein.

[0035] Figure 7 ELISA was used to detect the concentrations of TNF-α and IL-6 in the culture supernatant of spleen cells from immunized mice.

[0036] Figure 8 It is the neutralizing antibody titer of immune mouse serum against PoRV (G5 and G9 types) virus. DETAILED DESCRIPTION

[0037] Unless otherwise specified, the chemical reagents, biochemical reagents and materials used in the present invention can be obtained from commercial channels.

[0038] Example 1 FliC S.T -Construction, expression and identification of VP8 multi-epitope fusion protein

[0039] (1)FliC S.T -Construction and prediction of the structure of VP8 multi-epitope fusion protein

[0040] First, the PoRV VP8 protein (UniProt accession number: A0A1S5WJ26) and Salmonella typhimurium flagellin FliC were downloaded from the UniProt database (https: / / www.uniprot.org / ). S.T (UniProt accession number: P06179) complete amino acid sequence. Subsequently, the IEDB online B cell antigen epitope prediction tool (http: / / tools.immuneepitope.org / bcell) was used to predict the potential B cell neutralizing epitopes of the two proteins. On this basis, relying on the MEFA technology platform, FliC S.T As the backbone protein (the amino acid and nucleotide sequences are listed in SEQ ID NO.11 and SEQ ID NO.12, respectively), the selected dominant B cell neutralizing epitopes in VP8 protein (the amino acid and nucleotide sequences of the B cell neutralizing epitopes VP8-E1, VP8-E2, VP8-E3 and VP8-E4 are listed in SEQ ID NO.1 to 4 and SEQ ID NO.5 to 8, respectively) were used to replace FliC S.T The original epitopes with low immunogenicity and surface exposure on the backbone were used to construct FliC S.T -VP8 multi-epitope fusion protein (the corresponding amino acid and nucleotide sequences are shown in SEQ ID NO.9 and SEQ ID NO.10; the schematic diagram of its construction is shown in Figure 1 (as shown in A).

[0041] Next, the fusion protein sequence was homologously modeled using the Phyre2 platform. The generated structural models were comprehensively scored based on key indicators such as model confidence, sequence coverage, and homology (>90%), and the one with the highest score was selected as the final prediction model. The model was further analyzed for three-dimensional structure using PyMOL software. The results are as follows: Figure 1 B and 1C show that the insertion of exogenous VP8 epitope did not disrupt FliCS.T The overall folding of the skeleton; and the inserted epitopes are all located on the surface of the protein, which is conducive to its recognition and binding by B cell receptors.

[0042] (2)FliC S.T -PCR amplification of VP8 chimeric gene and VP8 gene

[0043] The plasmid pUC57-FliC was synthesized by Nanjing Qingke Biotechnology Co., Ltd. S.T -VP8 and pUC57-VP8 (GenBank accession number of VP8 gene: JQ011467.1, FliC S.T -VP8 gene and VP8 gene insertion site is between BamHI and SphI restriction sites of pUC57 plasmid) as amplification template, primers P7 (SEQ ID NO.13) and P8 (SEQ ID NO.14) were used to synthesize plasmid pUC57-FliC S.T -VP8 was PCR amplified; primer P9 (SEQ ID NO.15) and primer P10 (SEQ ID NO.16) were used to PCR amplify pUC57-VP8 to obtain the target chimeric gene fragment FliC S.T -VP8 and VP8 gene fragments.

[0044] The PCR reaction system (total volume 50 μL) was prepared as follows: template pUC57-FliC S.T -VP8 or pUC57-VP8 plasmid 4 μL, 5× Pfu enzyme buffer 10 μL, Pfu enzyme (Beijing Quanshijin Biotechnology, catalog number: AP221-01) 1 μL, dNTP mixture (2.5 mM) 4 μL, ddH2O 27 μL, P7 / P8 or P9 / P10 primers 2 μL each, the total reaction volume is 50 μL.

[0045] The specific PCR reaction conditions were set as follows: pre-denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 sec, annealing at 52°C for 30 sec, extension at 72°C for 2 min, and finally extension at 72°C for 7 min and storage at 4°C for termination.

[0046] The amplified products were detected by 1.0% agarose gel electrophoresis. Figure 2 As shown, FliC S.T -VP8 chimeric gene fragment showed a single clear band at about 1500bp, which was consistent with the expected fragment size. The target band was then recovered using the DNA gel recovery kit (Cat. No. DP214-03) from Tiangen Biochemical Technology Co., Ltd., and the concentration of the purified target gene fragment was determined using Nanodrop2000. S.TThe concentrations of the -VP8 chimeric gene fragment and VP8 gene fragment were 125 ng / μL and 136 ng / μL, respectively.

[0047] (3)pET-28a(+)-FliC S.T Construction of pET-VP8 recombinant plasmid and pET-28a(+)-VP8 recombinant plasmid

[0048] First, the purified FliC S.T The VP8 chimeric gene fragment was double-digested with BamHI-HF (NEB, Catalog No. R3136M) and SacI-HF (NEB, Catalog No. R3156M) at 37°C for 2.5 hours. The purified VP8 target gene fragment was double-digested with BamHI-HF (NEB, Catalog No. R3136M) and SacI-HF (NEB, Catalog No. R3138M); the pET-28a(+) plasmid was double-digested with the corresponding enzymes.

[0049] The enzyme digestion reaction system is configured as follows: 60 μL of gene fragment or plasmid, 2 μL each of BamHI-HF and SacI-HF / SalI-HF, 10 μL of 10× CutSmart Buffer, and 26 μL of ddH2O. Add these reagents sequentially to a PCR tube, mix thoroughly, and incubate in a 37°C water bath for 2.5 hours.

[0050] After enzyme digestion, the purified target gene fragment was ligated with the pET-28a(+) plasmid product and T4 DNA ligase (NEB, 10 U / μL) at 16°C overnight.

[0051] The ligation reaction system is as follows: 4 μL of pET-28a(+) plasmid digestion product, FliC S.T -VP8 or VP8 target gene digestion product 4 μL, T4 ligase 1 μL, 10×T4 ligase buffer 1 μL, total volume 10 μL.

[0052] The ligation product was then transformed into E. coli TOP10 competent cells, and 1 mL of resistance-free LB liquid medium was added. The cells were cultured at 37°C and 220 rpm for 2 hours. An appropriate amount of the bacterial suspension was spread onto a LB solid medium plate containing kanamycin sulfate and incubated at 37°C. 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.

[0053] Using overnight cultured bacteria as template, preliminary identification was performed by PCR. The electrophoresis results were as follows: Figure 3 As shown, pET-28a(+)-FliC S.TThe suspected positive clone of the VP8 recombinant plasmid showed a single band at approximately 1500bp, which was consistent with expectations. After PCR identification and confirmation of the positive clone, the sample was 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-28a(+)-FliC S.T -VP8 and pET-28a(+)-VP8.

[0054] (4)FliC S.T -Expression and identification of VP8 multi-epitope fusion protein and VP8 recombinant protein

[0055] The recombinant expression plasmid pET-28a(+)-FliC was successfully constructed. S.T -VP8 or pET-28a(+)-VP8 was transformed into the E. coli expression host strain BL21(DE3). A single positive clone was picked and inoculated into 5 mL of LB liquid medium containing 30 μg / mL kanamycin sulfate and cultured at 37°C and 220 rpm for 16 h. S.T -VP8 / BL21 recombinant expression bacteria were identified by PCR using primers P7 (SEQ ID NO.13) and P8 (SEQ ID NO.14), and pET-28a(+)-VP8 / BL21 recombinant expression bacteria were identified by PCR using primers P9 (SEQ ID NO.15) and P10 (SEQ ID NO.16) to confirm correctness. Next, the overnight culture of the recombinant strain was inoculated into 500 mL of LB medium containing kanamycin sulfate (final concentration of 30 μg / mL) at a ratio of 1:100 and cultured at 37°C with shaking until the OD 600 The value reached 0.6-0.8. Then IPTG was added at a final concentration of 1 mmol / L to induce expression for 4 hours. The induced bacteria were collected, ultrasonically disrupted, and the precipitate was collected after centrifugation. The inclusion body solution was fully dissolved at room temperature. 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. The results are as follows Figure 4 As shown, FliC is expressed at approximately 55 kDa. S.T -VP8 target band. The purified FliC was detected by BCA method. S.T -The concentration of VP8 multi-epitope fusion protein was 1.27 mg / mL; the concentration of VP8 recombinant protein was 1.56 mg / mL.

[0056] Example 2FliC S.T -Analysis of immunogenicity of VP8 multi-epitope fusion protein

[0057] (1) Mouse immunization

[0058] Twenty 7-week-old female BALB / c mice were randomly divided into 4 groups, with 5 mice in each group. The first group of mice was subcutaneously injected with 50 μg / mouse FliC S.T -VP8 recombinant protein; the second group of mice were subcutaneously injected with 50 μg / mouse VP8 recombinant protein (emulsified with an equal volume of complete Freund's adjuvant); the third group of mice were subcutaneously injected with 50 μg / mouse purified FliC S.T Recombinant protein (Pang et al., 2024); the fourth group, serving as the control group, received a subcutaneous injection of 100 μL of sterile 0.01 M PBS buffer (pH = 7.4). After the first immunization, booster immunizations were performed every 14 days for a total of three immunizations (days 0, 14, and 28). The second group received incomplete Freund's adjuvant on days 14 and 28. Blood was collected from the retroorbital vein before immunization (day 0) and on days 7, 14, 21, 28, 35, and 42 after immunization. Serum was separated and stored at -20°C. 14 days after the final immunization, mice were sacrificed by painless cervical dislocation, and terminal serum and spleen cells were collected for subsequent cytokine analysis.

[0059] (2) Western blot identification experiment

[0060] The purified VP8 recombinant protein sample was loaded into a 12% SDS-PAGE separation gel for electrophoresis and then transferred to a PVDF membrane. The membrane was blocked with a 5% skim milk solution at 4°C overnight. The PVDF membrane was then incubated with a 1:6000 dilution of VP8 recombinant protein immune serum and a 1:6000 dilution of FliC. S.T The membrane was incubated with VP8 recombinant protein immune serum (prepared in Example 2) at 4°C for 1.5 hours. After washing with PBST, a 1:10,000 dilution of HRP-labeled goat anti-mouse IgG (ABclonal, Catalog No. AS003) was added and incubated for another 1.5 hours at room temperature. After multiple PBST washes, the membrane was developed using ECL. After incubation in the dark for 1.5 minutes, the antigen-antibody binding signal was detected using a chemiluminescence imaging system.

[0061] The results are as follows Figure 5 As shown in the results, the purified VP8 protein can be specifically recognized by the sera of mice immunized with anti-VP8 recombinant protein and FliCS.T-VP8 recombinant protein, indicating that the FliCS.T-VP8 recombinant protein is not only successfully expressed, but also retains the immunogenicity of the inserted VP8 dominant epitope and has good antigenic epitope display function.

[0062] (3) Anti-VP8 specific IgG antibody detection

[0063] Purified VP8 recombinant protein was diluted to 5 ng / μL in ELISA coating buffer (0.05 M carbonate buffer, pH 9.6) and added to a 96-well plate at 100 μL / well. The plate was incubated at 37°C for 1 hour and then refrigerated at 4°C overnight. The next day, the plate was equilibrated at room temperature for 30 minutes, washed three times with PBST (0.05% Tween-20) (300 μL / well, 5 minutes each time), and then blocked with 200 μL / well of blocking buffer (10% skim milk powder / PBST) and incubated at 37°C for 1 hour. Washing was performed as above. Mouse immune serum was serially diluted in PBST (1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800). Each dilution was replicated in triplicate, and 100 μL / well of the reaction system was added. The plate was incubated at 37°C for 90 minutes. The plate was washed three times. Add 100 μL of 1:5000 diluted goat anti-mouse IgG-HRP (ABclonal, Catalog No. AS003) to each well and incubate at 37°C in the dark for 90 min. Wash the plate three times with PBST. Add 200 μL of TMB substrate to each well and develop color at 37°C in the dark for 30 min. Measure the optical density (OD) of each well at 650 nm.

[0064] The anti-VP8 antibody titer determination standard is: according to the serum sample OD 650 nm Value-blank control OD 650 nm The value ≥ 0.3 was recorded as positive, and then the value was multiplied by the highest dilution factor of the well, and finally the log 10 Calculate. The result is as follows Figure 6 As shown, from the 7th day after immunization, FliC S.T -VP8 multi-epitope fusion protein immunization group and VP8 recombinant protein and Freund's adjuvant immunization group can induce high titer anti-VP8 specific IgG, among which the antibody titer (log 10 ) were 3.87±0.05 and 3.93±0.03, respectively, while FliC S.T No corresponding antibodies were detected in the recombinant protein alone immunization group and PBS control group, indicating that FliC S.T -VP8 multi-epitope fusion protein has good immunogenicity.

[0065] (4) Detection of cytokines in spleen cell culture supernatant

[0066] On the 14th day after the last immunization, the mice in each group in step (1) were sacrificed by painless cervical dislocation. The spleen was removed under sterile conditions and placed in a sterile culture dish containing RPMI 1640 culture medium. The spleen was gently ground with a syringe piston with a needle until the tissue was completely dissociated. After the cell suspension was rinsed with RPMI 1640 three times, the cell pellet was collected by centrifugation at 1000r / min for 10min. 1× red blood cell lysis buffer was added, gently mixed and allowed to stand at room temperature for 5min until the red blood cells were completely lysed. Centrifuge again (1000r / min, 10min) and the supernatant was discarded. The cells were resuspended with sterile PBS and centrifuged repeatedly to completely remove the lysis buffer residue. The cells were then resuspended with RPMI 1640 culture medium containing 0.5% FBS and 1% penicillin-streptomycin double antibody, and the cell density was adjusted to 2.5×10 5 cells / mL. 2 mL of cell suspension was added to each 6-well culture plate and gently shaken using the "cross method" to ensure uniform plating. Purified VP8 recombinant protein was added to each well at a final concentration of 5 μg / mL and incubated in a 37°C, 5% CO2 incubator for 72 hours. After incubation, the supernatant was collected and the concentrations of IFN-γ (Xinbosheng, Catalog No. EMC101g) and IL-6 (Xinbosheng, Catalog No. EMC004) were determined according to the kit instructions.

[0067] The results are as follows Figure 7 As shown, FliC S.T In mice treated with the VP8 multi-epitope fusion protein, IFN-γ and IL-6 levels in the spleen cell culture supernatant were significantly higher than those in the PBS control group (P<0.05). These data indicate that the multi-epitope fusion protein can effectively induce strong cytokine secretion in addition to humoral immunity, demonstrating its excellent activation efficacy at the cellular immunity level.

[0068] (5) Neutralizing antibody detection

[0069] In a 96-well plate, add 100 μL of serum-free DMEM medium (containing 10 μg / mL trypsin) to each well. Add 100 μL of inactivated mouse immune serum to the first well, mix thoroughly, and then serially dilute in a 1:2 ratio to 1:2. 11 4 replicate wells were set up for each dilution. Subsequently, 100 μL of 200 TCID 50PoRV (G5 or G9 type) virus solution (laboratory storage) was incubated at 37°C for 1 hour to complete the antigen-antibody pre-incubation. After the incubation, MA104 cells (ATCC#CRL-2378.1) were inoculated and placed in an incubator containing 5% CO2 for overnight culture. The next day, the virus-serum mixture pre-incubated with the antibody was returned to a new 96-well plate (100 μL / well) in the order of the original wells. At the same time, normal cell control wells (8 wells, only cells were inoculated) and virus control wells (100 μL per well containing 200 TCID 50 PoRV (G5 or G9 type) virus solution, without immune serum). To evaluate the virus titer, 100 TCID 50 PoRV (G5 or G9) virus solution was diluted 10-fold (10 0 ~10 -3 ) with 100 μL per well, a total of 6 replicate wells, for virus regression testing. All wells were incubated at 37°C for 48 h.

[0070] After the culture was completed, the culture medium was discarded and the plate was washed 3 times with PBS; 150 μL of 80% acetone pre-cooled to -20°C was added to each well and fixed at 4°C for 30 minutes; the plate was washed again with PBS 3 times; blocked with 5% skim milk / PBST at 37°C for 1 hour, and the plate was washed with PBST. Subsequently, 200-fold diluted immune serum (100 μL / well) was added to each well and incubated in the dark at 37°C for 1 hour; after washing the plate with PBST, 500-fold diluted fluorescently labeled secondary antibody (100 μL / well) was added and incubated in the dark at 37°C for 1 hour; the plate was washed 3 times with PBST. Finally, the number of infected cells in each well was observed and recorded by fluorescence microscopy, and the neutralizing antibody titer was calculated based on this. The results showed that FliC S.T The neutralizing titers of serum from mice immunized with VP8 multi-epitope fusion protein against PoRV G5 and G9 were 1:2 respectively. 8 and 1:2 9 , which verified its good immunogenicity and showed its potential as a candidate epitope vaccine for PoRV ( Figure 8 ).

Claims

1. A dominant B cell antigen epitope peptide of porcine rotavirus VP8 protein or a combination thereof, characterized in that: The amino acid sequence of the B cell antigen epitope peptide or a combination thereof comprises at least one of SEQ ID NO.1 to SEQ ID NO.

4.

2. A nucleic acid molecule encoding the B cell antigen epitope peptide or a combination thereof according to claim 1, characterized in that: The DNA sequence of the nucleic acid molecule encoding the B cell antigen epitope peptide or a combination thereof comprises at least one of SEQ ID NOs. 5 to 8.

3. A FliC S.T -VP8 multi-epitope fusion protein, characterized in that The FliC S.T -VP8 multi-epitope fusion protein with Salmonella typhimurium flagellin FliC S.T As a skeleton protein, the dominant B cell antigen epitope peptide or its combination described in claim 1 replaces FliC S.T The B cell antigen epitope with low immunogenicity and exposed on the protein surface is constructed. Preferably, the FliC S.T -VP8 multi-epitope fusion protein is a protein that combines the dominant B cell antigen epitope peptide described in claim 1 with the Salmonella typhimurium flagellin FliC S.T In terms of the backbone, preferably, the amino acid sequence of the multi-epitope fusion protein is as shown in SEQ ID NO.

9.

4. Encoding the FliC according to claim 3 S.T -VP8 multi-epitope fusion protein nucleic acid molecule, characterized in that, The DNA sequence of the nucleic acid molecule is shown as SEQ ID NO.

10.

5. The FliC according to claim 3 S.T - A method for preparing a VP8 multi-epitope fusion protein, characterized in that: The following steps are involved: (1) Obtain FliC S.T -VP8 target gene fragment; (2) Place the FliC S.T -VP8 target gene fragment is connected with plasmid to obtain recombinant plasmid; (3) The recombinant plasmid is transferred into Escherichia coli for culture and induction of expression, and then the multi-epitope fusion protein is obtained after ultrasonic disruption and centrifugal separation and purification.

6. The FliC according to claim 5 S.T - A method for preparing a VP8 multi-epitope fusion protein, characterized in that: The nucleic acid molecule is obtained in step (1) by synthesizing the plasmid pUC57-FliC S.T -VP8 was used as a template for PCR amplification to obtain FliC S.T -VP8 target gene fragment, the primer sequences used in the PCR amplification are shown in SEQ ID NO.13 and SEQ ID NO.

14.

7. The FliC according to claim 5 S.T - A method for preparing a VP8 multi-epitope fusion protein, characterized in that: Step (2) is as follows: pET28a (+) plasmid and purified FliC obtained in step (1) are respectively S.T -VP8 target gene fragment was double-digested; the purified target gene fragment was mixed with the pET28a (+) plasmid vector after enzyme digestion, and T4 DNA ligase was added to carry out ligation reaction to obtain a ligation product; the ligation product was transformed into TOP10 competent cells, and the recombinant plasmid pET28a (+)-FliC was obtained after culture. S.T -VP8.

8. The dominant B cell antigen epitope peptide or a combination thereof according to claim 1, the nucleic acid molecule of the B cell antigen epitope peptide or a combination thereof according to claim 2, the FliC according to claim 3 S.T -VP8 multi-epitope fusion protein, FliC according to claim 4 S.T -Application of nucleic acid molecules of VP8 multi-epitope fusion protein in the preparation of drugs for preventing or treating porcine rotavirus infection.

9. The use according to claim 8, characterized in that The medicine includes a vaccine, an antibody or a diagnostic reagent.

10. A vaccine, neutralizing antibody or diagnostic reagent, characterized in that: The vaccine comprises the FliC according to claim 3 S.T -VP8 multi-epitope fusion protein; the neutralizing antibody comprises inducing the production of the multi-epitope fusion protein after immunizing an animal; the diagnostic reagent comprises FliC S.T -The immune serum of VP8 multi-epitope fusion protein specifically recognizes VP8 recombinant protein.

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