Porcine rotavirus VP7 multi-epitope fusion protein as well as preparation method and application thereof

Abstract

The invention discloses a porcine rotavirus VP7 protein conservative dominant B cell antigen epitope peptide or a combination thereof or a nucleic acid molecule and application thereof, and further discloses a porcine rotavirus VP7 protein multi-epitope fusion protein and a preparation method and application thereof. The multi-epitope fusion protein disclosed by the invention not only can induce high-level specific antibody response and cellular immune response aiming at the VP7 protein and effectively inhibit porcine rotavirus infection, but also has relatively high safety and stability. Compared with the traditional fusion protein, the multi-epitope fusion protein disclosed by the invention has remarkable advantages in the aspects of production cost, immune efficacy and the like, and a feasible porcine rotavirus prevention and control scheme is provided for the pig industry.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a porcine rotavirus VP7 protein multi-epitope fusion protein and a preparation method and application thereof. Background Art

[0002] Porcine rotavirus (PoRV) is one of the main pathogens causing acute gastroenteritis in piglets. Its clinical symptoms are mainly watery diarrhea, vomiting, dehydration and growth retardation. Since the virus was first isolated in the UK in 1974, it has become prevalent worldwide. Based on the group antigen specificity of the VP6 protein, rotavirus can be divided into 10 serogroups, A to J. Among them, RVA, RVB, RVC, RVE and RVH are the main ones that infect pigs. RVA has attracted much attention due to its strongest pathogenicity and widest prevalence. The genotyping of RVA is mainly based on the phylogenetic analysis of the VP4 (P type) and VP7 (G type) genes. Currently, 35 G types and 50 P types have been identified. Epidemiological monitoring shows that common G types include G1 to G6 and G9, while common P types include P[6], P[7], P[8], P

[11] , P

[13] , P

[23] and P

[32] . In pig farming, G9, G5, and G3 are the main epidemic types, accounting for 68.7% of cases, while P

[13] and P

[23] constitute 82.1% of the main epidemic P types; globally, G5P[7] is the most prevalent genotype combination. Since PoRV was first detected in my country in 1983, the virus has spread throughout the country. Studies have shown that in the detection of 25,768 diarrhea samples collected from various regions, the average positive rate of PoRV was as high as 86.09%, and the positive rate of pig farms detected was 80%, which fully proves that it poses a serious threat to my country's breeding industry.

[0003] PoRV belongs to the Reoviridae family. The mature virus has an icosahedral symmetry, revealing a typical cartwheel shape under electron microscopy, with a diameter of approximately 75 nm. The virion consists of a three-layer capsid: the innermost capsid is a hexagonal prism composed of the VP2 protein, which encapsulates the viral genomic RNA; the middle capsid is composed of 32 VP6 proteins, which determine the viral group antigenicity; and the outer capsid, composed of VP7 and VP4, not only determines the viral G-type but also plays a key role in particle assembly, elicits neutralizing antibodies, and is highly conserved. The PoRV genome consists of 11 segmented double-stranded RNAs, totaling approximately 18.2 kb in length, encoding six structural proteins (VP1-VP4, VP6, and VP7) and six nonstructural proteins (NSP1-NSP6). VP1, VP2, and VP3 constitute the viral core. VP1 possesses RNA polymerase activity; VP2 binds to the genomic RNA to maintain structural stability; and VP3 prevents mRNA degradation and promotes translation. As receptor-binding proteins, VP5 and VP8 formed after VP4 is treated with trypsin play an enhancing role in the virus invasion and attachment processes respectively. Among them, VP8 contains the main neutralizing antigen and becomes an important target for vaccine development; VP6 not only participates in the maintenance of the virus structure, but also serves as the basis for grouping; VP7 and VP4 together constitute the virus's outer shell, determine the G type and induce the production of neutralizing antibodies.

[0004] Currently, prevention and control strategies for PoRV infection primarily include nutritional regulation, drug therapy, biosafety measures, and vaccination. Due to the lack of targeted antiviral drugs, nutritional intervention can, to a certain extent, reduce viral loads and enhance piglet resistance. Clinically, symptomatic supportive treatment with oral rehydration salts, tannic acid protein, and broad-spectrum antibiotics is commonly used. Biosafety measures require isolation of sick pigs, strict personnel management, regular disinfection, and optimized husbandry. However, these measures only provide temporary relief and are insufficient to address the increased virulence and antigenic drift associated with the continued evolution of PoRV. In contrast, vaccination, due to its cost-effectiveness and long-lasting protection, is considered one of the most effective means of preventing PoRV infection. Currently, vaccines primarily include traditional inactivated vaccines, live attenuated vaccines, and newer genetically engineered vaccines. However, due to the diverse serotypes, volatile viral transmission, frequent interspecies transmission, and genetic rearrangement, there is a lack of effective cross-protection between different genotypes, and the effectiveness of existing vaccines remains to be improved. Therefore, the development of safer, more effective, and broadly protective vaccines is urgent. In recent years, bacterial flagellin has attracted widespread attention as a novel immune adjuvant. It can simultaneously induce humoral immunity and cellular immunity, overcome oral tolerance, and significantly promote the secretion of mucosal pro-inflammatory factors. It also has high structural plasticity (i.e., exogenous genes can be inserted into multiple functional regions without destroying their natural conformation), thereby synergistically enhancing the immune adjuvant effect in multiple ways. It has been widely used in the research and development of vaccines against pathogens such as bacteria, viruses and parasites.

[0005] Although flagellin adjuvants have demonstrated significant advantages in preclinical studies, their practical application currently faces two key challenges: first, the paradox of immunogenicity and safety. As a key virulence factor for some pathogens, flagellin exhibits high immunogenicity, which can lead to excessive immune and inflammatory responses, potentially damaging the body. Furthermore, pre-existing neutralizing antibodies can induce immune tolerance, further contributing to vaccine failure. Therefore, balancing flagellin's immunogenicity and safety is a key challenge in its application. Second, there is the conflict between structural stability and functional activity. Flagellin's hypervariable region has been modified to reduce immunogenicity by truncation or insertion of exogenous genes, thereby optimizing its adjuvant function. However, such modifications can disrupt the structure of the TLR5-binding domain, thereby weakening its adjuvant potency. Therefore, maintaining flagellin's structural stability and functional activity during genetic modification remains a technical challenge in development. Therefore, while bacterial flagellin has broad potential as an immune adjuvant, its widespread application and further optimization still require addressing several technical challenges, including immunogenicity, safety, structural stability, and functional activity. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a multi-epitope fusion protein and its preparation method and application, wherein the multi-epitope fusion protein is derived from the dominant B cell neutralizing epitope of the VP7 protein of PoRV, based on the flagellin FliC of Salmonella typhimurium. S.T It is used as an adjuvant, and through unique fusion protein design and autologous adjuvant effect, the immune effect of multi-epitope fusion protein is greatly enhanced.

[0007] Technical solution: The present invention provides a conserved dominant B cell antigen epitope peptide of porcine rotavirus VP7 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.

[0008] The present invention also provides a 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.

[0009] The present invention also provides a multi-epitope fusion protein, wherein the multi-epitope fusion protein is a Salmonella typhimurium flagellin F1iC 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 was constructed.

[0010] The multi-epitope fusion protein is a protein that sequentially connects the dominant B cell antigen epitope peptides to the Salmonella typhimurium flagellin FliC S.T On the skeleton.

[0011] Wherein, the amino acid sequence of the multi-epitope fusion protein is shown in SEQ ID NO.9.

[0012] The present invention also provides a nucleic acid molecule encoding the multi-epitope fusion protein, and the DNA sequence of the nucleic acid molecule is shown as SEQ ID NO.10.

[0013] The present invention also provides a method for preparing the multi-epitope fusion protein, comprising the following steps:

[0014] (1) obtaining a nucleic acid molecule encoding the dominant B cell antigen epitope peptide or a combination thereof;

[0015] (2) connecting the nucleic acid molecule described in step (1) with the plasmid to obtain a recombinant plasmid;

[0016] (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.

[0017] The present invention also provides the use of the dominant B cell antigen epitope peptides or their combination, the nucleic acid molecules of the B cell antigen epitope peptides or their combination, the multi-epitope fusion protein, and the nucleic acid molecules of the multi-epitope fusion protein in the preparation of drugs for preventing and treating porcine rotavirus infection.

[0018] Wherein, the medicine includes a vaccine, an antibody or a diagnostic reagent.

[0019] The present invention also provides a vaccine or antibody, wherein the vaccine comprises the multi-epitope fusion protein; and the antibody is induced after immunizing an animal with the multi-epitope fusion protein.

[0020] Furthermore, the present invention also includes FliC S.T -VP7 multi-epitope fusion protein. The FliC of the present invention S.T -VP7 multi-epitope fusion protein has an amino acid sequence as shown in SEQ ID NO.11 and a nucleotide sequence as shown in SEQ ID NO.12. S.T As a skeleton protein; replace FliC with the VP7 dominant B cell neutralizing epitope as shown in SEQ ID NO.1-4 and the nucleotide sequence as shown in SEQ ID NO.5-8 S.T Construction of B cell antigen epitopes with low immunogenicity and exposed on the protein surface.

[0021] Furthermore, the present invention also includes FliC S.T -VP7 multi-epitope fusion protein, the amino acid sequence of which is shown in SEQ ID NO.9.

[0022] The present invention also includes a nucleic acid molecule encoding the multi-epitope fusion protein, and the DNA sequence of the nucleic acid molecule is shown in SEQ ID NO.10.

[0023] Furthermore, the present invention also provides the FliC S.T -The preparation method of VP7 multi-epitope fusion protein specifically comprises the following steps:

[0024] (1) Synthesize plasmid pUC57-FliC S.T -VP7 was used as the template for PCR amplification to obtain FliC S.T -VP7 target gene fragment;

[0025] (2) The pET-28a(+) plasmid and purified FliC S.T-VP7 target gene fragment was subjected to double enzyme digestion reaction, and the purified target gene fragment was mixed with pET-28a(+) plasmid product and T4 DNA ligase to obtain a ligation product, which was then transformed into TOP10 competent cells and cultured to obtain the recombinant plasmid pET-28a(+)-FliC S.T -VP7;

[0026] (3) The recombinant expression plasmid pET-28a(+)-FliC S.T -VP7 was transformed into Escherichia coli BL21 (DE3) for culture and induced expression, and then ultrasonically broken and centrifuged for purification to obtain the FliC S.T -VP7 multi-epitope fusion protein.

[0027] Furthermore, in step (1), the sequences of primers P7 and P8 used in the PCR amplification process are shown in SEQ ID NOs. 13-14.

[0028] 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-HF and SacI-HF, 10 μL of 10×CutSmart Buffer, and 6 μL of ddH2O. The above reagents are added to the PCR tube in sequence, mixed thoroughly, and reacted in a constant temperature water bath at 37°C for 2.5 hours.

[0029] The present invention also provides a PoRV multi-epitope vaccine, wherein the PoRV multi-epitope vaccine comprises the FliC S.T -VP7 multi-epitope fusion protein.

[0030] Furthermore, the FliC S.T -VP7 multi-epitope fusion protein can induce the production of high levels of neutralizing antibodies against PoRV after being used to immunize mice.

[0031] Furthermore, the FliC S.T -The immune serum of VP7 multi-epitope fusion protein can recognize VP7 recombinant protein.

[0032] Beneficial effect: The present invention innovatively uses the flagellin FliC of Salmonella typhimurium S.T As a backbone protein, and based on the multi-epitope fusion antigen technology (MEFA) platform, the dominant B cell neutralizing epitope in PoRV VP7 protein was precisely embedded into FliC through optimized design. S.T The hypervariable regions of porcine rotavirus were used to construct a multi-antigen fusion protein with both broad spectrum and high efficiency, providing a more ideal technical solution for the prevention and control of PoRV infection. Specifically, it includes the following aspects:

[0033] (1) Self-adjuvant effect: Using Salmonella typhimurium flagellin FliC with excellent adjuvant effect S.T As the backbone, the VP7 dominant B cell neutralizing epitope of PoRV was firstly combined with FliC S.T Functional fusion expression of FliC S.T As an immunostimulatory molecule, it can significantly activate the host's innate and adaptive immune responses, allowing the multi-epitope fusion protein to stimulate cellular immune responses while inducing the production of neutralizing antibodies. S.T The skeleton can significantly enhance its immune protection effect against PoRV. The multi-epitope fusion protein can achieve significant immune effect without additional adjuvants, reducing production costs and simplifying immunization operations.

[0034] (2) Enhanced immune protection: The multi-epitope fusion protein of the present invention not only induces strong humoral immunity but also stimulates effective cellular immunity, thereby enhancing the protective effect of the multi-epitope vaccine. The fusion protein can effectively induce neutralizing antibodies against PoRV and enhance the cellular immune response, providing comprehensive immune protection for the piglet population.

[0035] In summary, the multi-epitope fusion protein FliC prepared by the present invention S.T -VP7 as a PoRV-based VP7 dominant B cell neutralizing epitope and FliC S.T This fusion protein with an auto-adjuvant effect combines advanced techniques such as immunodominant epitopes, multi-epitope fusion protein technology, and genetic engineering to enhance vaccine specificity and protective efficacy. The vaccine prepared by this invention not only induces a high-level humoral immune response, effectively neutralizing PoRV infection, but also induces a robust cellular immune response. Compared with traditional vaccines, this vaccine offers significant advantages in production cost, immune efficacy, and durability of protection, providing a practical and feasible PoRV prevention and control solution for the swine industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0037] Figure 1 For FliC S.T -Schematic diagram of the construction of VP7 multi-epitope fusion protein and structure prediction diagram, where E1 to E4 represent the dominant B cell neutralizing epitopes of VP7 protein of PoRV. Figure 1 A is FliC S.T-Schematic diagram of the construction of VP7 multi-epitope fusion protein, Figure 1 B is FliC S.T -Prediction of the secondary structure of VP7 multi-epitope fusion protein; Figure 1 C is FliC S.T -Prediction of the tertiary structure of VP7 multi-epitope fusion protein.

[0038] Figure 2 For FliC S.T -PCR amplification results of VP7 chimeric gene, where lane M is 2K Plus II DNA Marker; lane 1 is FliC S.T -VP7 chimeric gene; lane 2 is the negative control.

[0039] Figure 3 For the recombinant expression plasmid pET-28a(+)-FliC S.T -VP7 PCR identification electrophoresis diagram, where lane M is 2KPlus II DNA Marker; lane 1 is the recombinant expression plasmid pET-28a(+)-FliC S.T -PCR amplification results of VP7; lane 2 is the negative control.

[0040] Figure 4 For purified FliC S.T - SDS-PAGE of VP7 multi-epitope fusion protein, where M is the protein molecular weight standard; lane 1 is the purified FliC S.T -VP7 multi-epitope fusion protein; Lane 2 is purified FliC S.T recombinant protein; lane 3 is the purified VP7 recombinant protein.

[0041] Figure 5 This is a Western blot analysis of the purified VP7 recombinant protein. Figure 5 A is the reaction diagram of VP7 recombinant protein and anti-VP7 polyclonal antibody; Figure 5 B is VP7 recombinant protein and anti-FliC S.T - VP7 polyclonal antibody reactivity graph.

[0042] Figure 6 ELISA for detecting FliC S.T -Specific anti-VP7 antibody levels in the serum of mice immunized with VP7 multi-epitope fusion protein.

[0043] Figure 7 ELISA was used to detect the concentrations of IFN-γ and IL-6 in the culture supernatant of spleen cells from immunized mice.

[0044] Figure 8It is the neutralizing antibody titer of immune mouse serum against PoRV (G5 type) virus. DETAILED DESCRIPTION

[0045] 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0047] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents of this specification shall prevail.

[0048] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0049] The terms “include,” “including,” “have,” “contain,” etc. used in the present invention are open-ended terms, meaning including but not limited to.

[0050] Unless otherwise specified, the chemical reagents, biochemical reagents and materials used in the present invention can be obtained from commercial sources. S.T -Construction, expression and identification of VP7 multi-epitope fusion protein

[0051] (1)FliC S.T -Structure prediction and construction of VP7 multi-epitope fusion protein

[0052] By logging into the UniProt database (https: / / www.uniprot.org / ), we obtained the VP7 protein of PoRV (accession number: P32546) and FliC S.T The complete amino acid sequence of protein (accession number: P06179) in FASTA format was obtained. Then, the B cell epitopes of the two proteins were predicted using the B cell antigen epitope online prediction tool (http: / / tools.immuneepitope.org / bcell). The specific prediction parameters were as follows: the threshold of epitope probability was set to 0.5; at least seven consecutive amino acid residues with an epitope probability score exceeding 0.5 were selected as a putative B cell epitope. Then, the MEFA technology was used to construct the FliC S.T -VP7 multi-epitope fusion protein, the construction diagram of which is shown in Figure 1 As shown in A.

[0053] Specifically, the flagellin FliC of Salmonella typhimurium S.T (its amino acid sequence is shown in SEQ ID NO.11, and its nucleotide sequence is shown in SEQ ID NO.12) as a scaffold protein, and the VP7 dominant B cell neutralizing epitopes (the amino acid sequences of VP7-E1, VP7-E2, VP7-E3 and VP7-E4 are shown in SEQ ID NOs.1 to 4, and the nucleotide sequences of VP7-E1, VP7-E2, VP7-E3 and VP7-E4 are shown in SEQ ID NOs.5 to 8) are used to replace the B cell epitopes with low immunogenicity and exposed on the protein surface in the scaffold protein, thereby constructing FliC S.T -VP7 multi-epitope fusion protein (its amino acid sequence is shown in SEQ ID NO.9, and its nucleotide sequence is shown in SEQ ID NO.10).

[0054] Subsequently, the multi-epitope fusion protein was structurally modeled using the Phyre2 portal, and the constructed models were scored using key parameters such as credibility, linear coverage, and homology, thereby screening out the FliC with the highest score (credibility > 95%, linear coverage > 92%, homology > 95%). S.T -VP7 protein model was used as the final model. Its structure was analyzed using PyMOL software. The results are as follows Figure 1 B and 1C show that the inserted foreign B cell epitope does not disrupt FliC S.T The native conformation of the scaffold protein and the inserted epitopes are exposed to FliC S.T -The surface of VP7 multi-epitope fusion protein helps to achieve the expected antigen presentation effect.

[0055] (2)FliC S.T- PCR amplification of VP7 and VP7 genes

[0056] The plasmid pUC57-FliC was synthesized by Nanjing Qingke Biotechnology Co., Ltd. S.T -VP7 and pUC57-VP7 (where FliC S.T -VP7 gene and VP7 gene (GenBank accession number: MH137265.1) were inserted between BamHI and SphI restriction sites of pUC57 plasmid) as amplification templates, and primers P7 (SEQ ID NO.13) and P8 (SEQ ID NO.14); primers P9 (SEQ ID NO.15) and P10 (SEQ ID NO.16) were used for PCR amplification, and FliC was successfully obtained. S.T -VP7 and VP7 gene fragments.

[0057] The PCR reaction system was configured as follows: template pUC57-FliC S.T -VP7 or pUC57-VP7 plasmid 4μL, 5×Pfu enzyme buffer 10μL, Pfu enzyme (Beijing Quanshijin Biotechnology, product number: AP221-01) 1μL, dNTP mixture (2.5mM) 4μL, ddH2O 27μL, P7 / P8 or P9 / P10 primers 2μL each, the total reaction volume is 50μL.

[0058] The 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 termination at 72°C for 7 min and storage at 4°C.

[0059] PCR amplification results are shown in Figure 2 , FliC can be seen at about 1530 bp S.T -VP7 target band, consistent with the expected result. The target gene fragment was recovered using the DNA gel recovery kit (Cat. No.: DP214-03) from Tiangen Biochemical Technology Co., Ltd.

[0060] (3) Construction of pET-28a(+)-FliCs.r-VP7 and pET-28a(+)-VP7 recombinant plasmids

[0061] First, the purified FliC S.TThe VP7 target gene fragment was double-digested with BamHI-HF (NEB, Cat. No. R3136M) and SacI-HF (NEB, Cat. No. R3156M), respectively. The purified VP7 target gene fragment was double-digested with NheI-HF (NEB, Cat. No. R3131M) and SalI-HF (NEB, Cat. No. R3138M), respectively. The pET-28a(+) plasmid was double-digested with the corresponding endonucleases. The digestion reaction system was as follows: 60 μL of gene fragment or plasmid, 2 μL each of BamHI-HF / NheI-HF and SacI-HF / SalI-HF, 10 μL of 10× CutSmart Buffer, and 26 μL of ddH2O. These reagents were added sequentially to a PCR tube, mixed thoroughly, and incubated in a 37°C water bath for 2.5 hours.

[0062] After enzyme digestion, the purified target gene fragment was mixed with the pET-28a(+) plasmid product and T4 DNA ligase and ligated at 16°C overnight.

[0063] The ligation reaction system is as follows: 4 μL of pET-28a(+) plasmid digestion product, FliC S.T -VP7 or VP7 target gene digestion product 4 μL, T4 ligase 1 μL, 10×T4 ligase buffer 1 μL, total volume 10 μL.

[0064] The ligation product was then transformed into 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 plate containing LB solid medium 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.

[0065] Using overnight cultured bacteria as template, preliminary identification was performed by PCR. The results were as follows: Figure 3 As shown, the suspected positive clone showed the target band at approximately 1533bp, 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 -VP7 and pET-28a(+)-VP7.

[0066] (4)FliC S.T -Expression and identification of VP7 and VP7 recombinant protein

[0067] The recombinant expression plasmid pET-28a(+)-FliC S.T-VP7 or pET-28a(+)-VP7 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 with a shaker at 220 r / min for 16 h. S.T -VP7 / BL21 recombinant expression bacteria were identified by PCR using primers P7 (SEQ ID NO.13) and P8 (SEQ ID NO.14), and pET-28a(+)-VP7 / 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, a clear band was shown at about 55 KDa. The purified FliC was determined by BCA method. S.T -The concentration of VP7 multi-epitope fusion protein is 1.25 mg / mL; the concentration of VP7 recombinant protein is 0.68 mg / mL.

[0068] Example 2FliC S.T -Analysis of immunogenicity of VP7 multi-epitope fusion protein

[0069] (1) Mouse immunization

[0070] Twenty 7-week-old BALB / c female mice were randomly divided into 4 groups (5 mice in each group). The first group of mice were subcutaneously injected with 50 μg of FliC S.T -VP7 multi-epitope fusion protein; the second group of mice were subcutaneously immunized with the recombinant expressed VP7 protein mixed with an equal volume of Freund's complete adjuvant, with an immunization dose of 50 μg per mouse; the third group of mice were subcutaneously injected with 50 μg of purified FliC S.Trecombinant protein (Pang et al., 2024); the fourth group served as the control group and received a subcutaneous injection of 100 μL of autoclaved 0.01 M PBS buffer (pH = 7.4). The specific immunization procedure was as follows: after the first immunization, booster immunizations were performed every two weeks, and the second group used incomplete Freund's adjuvant for the second and third immunizations. Blood was collected from the retroorbital vein before immunization (0 day) 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, the mice were sacrificed by painless cervical dislocation, and serum and spleen cells were collected for detection of inflammatory cytokines.

[0071] (2) Western blot verification experiment

[0072] The purified VP7 recombinant protein sample was loaded onto a 12% separation gel for SDS-PAGE. After electrophoresis, the protein was transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at 4°C overnight. The PVDF membrane was then incubated with anti-VP7 recombinant protein and FliC diluted 1:6000 in 5% skim milk. S.T -VP7 immune mouse serum was incubated at 4°C for 1.5 hours, then washed with PBST, and then added with 1:10000 diluted goat anti-mouse IgG-HRP secondary antibody (ABclonal, catalog number: AS003) and incubated at room temperature for 1.5 hours. Finally, after multiple washes with PBST, ECL luminescent solution was used to react for 90 seconds in the dark, and the specific binding of the target protein to each antibody was detected by chemiluminescence imaging system.

[0073] The results are as follows Figure 5 As shown, the purified VP7 protein can be detected by anti-VP7 recombinant protein and FliC S.T -VP7 recombinant protein was specifically recognized by the sera of immunized mice, indicating that FliC S.T -VP7 recombinant protein was not only successfully expressed, but also retained the immunogenicity of the inserted VP7 dominant epitope and had good antigenic epitope display function.

[0074] (3) Anti-VP7 specific IgG antibody detection

[0075] Purified VP7 recombinant protein was diluted in ELISA coating buffer (0.05 M carbonate buffer, pH 9.6) to a final concentration of 5 μg / mL and added to a 96-well plate at 100 μL / well. After incubation at 37°C for 1 hour, the plate was transferred to a 4°C refrigerator overnight. The next day, the plate was equilibrated at room temperature for 30 minutes and washed three times with PBST buffer (300 μL / well for 5 minutes each time). The plate was gently tapped to remove any residual liquid. Then, 200 μL / well of 10% skim milk / PBST was added and blocked at 37°C for 1 hour. Unbound material was removed by the above washing procedure. Immune serum samples were serially diluted in PBST (1:200 to 1:12800, specifically 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800). Each step was replicated in triplicate, and 100 μL / well was added to the reaction system. After incubation at 37°C for 90 minutes, complete three PBST washes. Add HRP-labeled goat anti-mouse IgG secondary antibody (1:5000 dilution / PBST), 100 μL / well, and react at 37°C in the dark for 90 minutes. After three PBST washes, add 200 μL TMB substrate solution to each well and develop at 37°C in the dark for 30 minutes. Immediately use an enzyme reader to read the absorbance value at a wavelength of 650 nm. The titer of the anti-VP7 antibody is determined as follows: according to the OD value of the serum sample 650 Value-blank control OD 650 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, FliC S.T -VP7 multi-epitope fusion protein immunization group and VP7 recombinant protein and Freund's adjuvant immunization group could induce high levels of anti-VP7 protein specific IgG antibodies from the 7th day, and the final antibody titer (log 10 ) were 3.65±0.13 and 3.70±0.11 respectively; while FliC S.T No corresponding antibodies were detected in the recombinant protein alone immunization group and the PBS control group. S.T -VP7 multi-epitope fusion protein has good immunogenicity. (4) Detection of cytokines in spleen cell culture supernatant

[0076] 14 days after the last immunization, the mice were killed by painless cervical dislocation, and the spleen was then aseptically removed and placed in a culture dish containing RPMI 1640 medium. Use a sterile syringe piston to gently grind until the tissue is completely dissociated. Subsequently, the cell sieve was rinsed 3 times with RPMI1640 medium, and the resulting cell suspension was collected into a 15 mL centrifuge tube and centrifuged at 1000 r / min for 10 min to collect the cell pellet. Next, 1× red blood cell lysis buffer (Beijing Solebold Technology Co., Ltd., catalog number: R1010) was added, gently mixed with a pipette, and allowed to stand for 5 minutes until the red blood cells were completely lysed. Then, centrifuged at 1000 r / min for 10 minutes, and the supernatant was discarded. Then, an appropriate amount of sterile PBS was added to slowly resuspend the cells, and the centrifugation was repeated at 1000 r / min for 10 minutes to remove residual PBS. Finally, the cell pellet was washed again with RPMI 1640 medium to remove residual PBS and tissue debris in the cells, and then resuspended in RPMI 1640 medium containing 1% penicillin-streptomycin and 0.5% FBS to adjust the cell concentration to 2.5 × 10 5 2mL of lymphocyte suspension was evenly distributed in a 6-well cell culture dish, and the cell plate was gently shaken using the "cross method" to ensure uniform cell density in each well. 5μg / mL of purified VP7 recombinant protein was added to each well to stimulate spleen cells, and the supernatant was collected after culturing in an incubator at 37°C and 5% CO2 for 72 hours. According to the instructions of the kit, ELISA was used to detect the concentrations of cytokines IFN-y (Shenzhen Xinbosheng Biotechnology Co., Ltd., Product No.: EMC101g) and IL-6 (Shenzhen Xinbosheng Biotechnology Co., Ltd., Product No.: EMC004) in the supernatant of spleen cell culture. The results are as follows Figure 7 As shown, FliC S.T -The levels of IFN-γ and IL-6 in the spleen cells of mice in the VP7 multi-epitope fusion protein immunization group were significantly higher than those in the PBS control group, indicating that the multi-epitope fusion protein can effectively stimulate the immune mice to produce high levels of cytokines and induce a good cellular immune response after immunization.

[0077] (5) Neutralizing antibody detection

[0078] In a 96-well plate, add 100 μL of serum-free DMEM containing 10 μg / mL trypsin to each well, add 100 μL of inactivated serum to the first well, mix well, and then transfer 100 μL to each well in turn, at a ratio of 1:2, 1:2. 2 , 1:2 3 ,......,1:2 11 Perform gradient dilution, and set up 4 replicate wells for each dilution. 50, 100 μL per well) was added to the cell plate, incubated at 37°C for 1 hour, and then inoculated with MA104 cells (ATCC#CRL-2378.1) and cultured overnight in a cell culture incubator containing 5% CO2. Subsequently, the virus solution co-incubated with immune serum was inoculated into 96-well cell plates in sequence, with 100 μL added to each well. Normal cell control wells (8 wells) and virus control wells (100 μL per well containing 200 TCID 50 In addition, DMEM containing 2% FBS (Gibco, catalog number: 6125197) was used to maintain the virus containing 100 TCID 50 The virus solution was diluted 10-fold (dilution: 100, 10, 10 -1 , 10 -2 , 10 -3 ), add 100 μL to each well, set up 6 replicate wells, and perform virus regression test. After culturing in a 37°C constant temperature incubator for 48 hours, discard the culture medium and wash the plate 3 times; add 150 μL of pre-cooled 80% acetone to each well, fix at 4°C for 30 minutes, then wash the plate 3 times with sterile PBS, add 37°C skim milk to block for 1 hour, and then wash the plate with PBST. Next, add 200-fold diluted inactivated immune serum to each well, incubate at 37°C in the dark for 1 hour, add 500-fold diluted fluorescent secondary antibody after washing, incubate at 37°C in the dark for 1 hour, and then wash the plate 3 times with PBST. Finally, observe and record the number of cell fluorescence under a fluorescence microscope, and calculate the neutralizing antibody titer based on the data of each well. The results are as follows Figure 8 As shown, FliC ST- The neutralizing antibody titer of the VP7 multi-epitope fusion protein immunization group was 1:2 9 , indicating that the fusion protein has good immunogenicity and can be used as a potential candidate epitope vaccine for PoRV.

Claims

1. A conserved dominant B cell antigen epitope peptide of porcine rotavirus VP7 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 multi-epitope fusion protein, characterized in that: The multi-epitope fusion protein is based on the flagellin FliC of Salmonella typhimurium 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 was constructed.

4. The multi-epitope fusion protein according to claim 3, characterized in that The multi-epitope fusion protein is a protein that sequentially connects the dominant B cell antigen epitope peptides described in claim 1 to the flagellin FliC of Salmonella typhimurium. S.T On the skeleton.

5. The multi-epitope fusion protein according to claim 3, characterized in that The amino acid sequence of the multi-epitope fusion protein is shown in SEQ ID NO.

9.

6. A nucleic acid molecule encoding the multi-epitope fusion protein according to any one of claims 3 to 5, characterized in that: The DNA sequence of the nucleic acid molecule is shown in SEQ ID NO.

10.

7. The method for preparing the multi-epitope fusion protein according to any one of claims 3 to 5, characterized in that: The following steps are involved: (1) obtaining a nucleic acid molecule encoding the dominant B cell antigen epitope peptide or a combination thereof as described in claim 1; (2) connecting the nucleic acid molecule described in step (1) with the plasmid to obtain a recombinant plasmid; (3) The recombinant plasmid is transferred into Escherichia coli for culture, and after induction of expression, it is broken by ultrasonication and purified by centrifugation to obtain the multi-epitope fusion protein.

8. Use of 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 multi-epitope fusion protein according to any one of claims 3 to 5, and the nucleic acid molecule encoding the multi-epitope fusion protein according to claim 6 in the preparation of a drug for preventing and 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 or antibody, characterized in that The vaccine comprises the multi-epitope fusion protein according to any one of claims 3 to 5; the antibody comprises the antibody induced by immunizing an animal with the multi-epitope fusion protein.

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