Multi-epitope fusion protein based on porcine rotavirus VP7 and VP8 double targets as well as preparation method and application of multi-epitope fusion protein

Abstract

The invention discloses a multi-epitope fusion protein based on porcine rotavirus VP7 and VP8 double targets as well as a preparation method and application of the multi-epitope fusion protein. According to the invention, salmonella typhimurium flagellin FliCS.T is taken as a skeleton, dominant neutralizing epitopes of VP7 and VP8 are accurately integrated, and the multi-epitope fusion antigen FliCS.T-VP7 + VP8 is constructed. The fusion protein has two immunological advantages: an FliCS.T skeleton has TLR5 adjuvant activity, so that innate immunity and adaptive immunity of a host can be efficiently activated; key neutralizing epitopes of porcine rotaviruses VP7 and VP8 are synchronously presented through a multi-epitope strategy, and high-titer specific neutralizing antibody and potent cellular immune response aiming at two structural proteins can be induced in vivo. The invention provides the multi-epitope fusion antigen of the porcine rotavirus, which integrates high immunogenicity and broad-spectrum neutralization protection, and a feasible technical scheme is provided for prevention and control of the porcine rotavirus.

Description

Technical Field

[0001] The present invention relates to the field of vaccine biotechnology, and in particular to a multi-epitope fusion protein based on the dual targets of porcine rotavirus VP7 and VP8, and a preparation method and application thereof. Background Art

[0002] Porcine rotavirus (PoRV) is a major pathogen facing the global swine industry, posing a particularly severe threat to suckling piglets. Epidemiological surveys show that 40% to 60% of pig herds test positive for PoRV, with over 10% of piglets displaying clinical symptoms. The harmfulness of PoRV is significantly exacerbated when co-infected with pathogens such as porcine epidemic diarrhea virus (PEDV) or enterotoxigenic Escherichia coli (ETEC). PoRV primarily invades the mature epithelial cells of the small intestinal villi of piglets, causing villous atrophy and crypt hyperplasia, leading to nutrient absorption impairment and diarrhea lasting 3-4 days. Although the virus causes relatively low direct mortality, it significantly slows piglet growth, reduces feed utilization, and affects weaning weight and survival rate. PoRV exists in five serogroups: A, B, C, E, and H, with serogroup A being the most prevalent. Notably, the detection rate of PoRV type C has increased rapidly in recent years. In my country, the infection rate in piglets under three days of age has exceeded 30%, indicating that PoRV type C has become a significant pathogen of enteritis in neonatal piglets. The virus is highly stable in the environment and requires effective inactivation with chlorine-containing disinfectants. Diagnosis primarily relies on reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), and careful consideration must be given to excluding co-infection with pathogens such as PEDV and ETEC.

[0003] The PoRV genome consists of 11 double-stranded RNA segments, approximately 18.2 kb in length, encoding six structural proteins (VP1–VP4, VP6, and VP7) and six nonstructural proteins (NSP1–NSP6). VP7 and VP4 together form the smooth outer capsid of the virus, determining the G subtype and inducing the production of neutralizing antibodies. VP4 is cleaved by trypsin to produce the VP8 subunit, which binds to sialic acid or HBGAs on the host cell surface, mediating viral adsorption. Compared to VP5, VP8 has higher immunogenicity, better stability, and expression yield. Antibodies against VP8 not only neutralize the same virus type but also provide cross-protection against different P subtypes. Its conserved receptor-binding region is an important target for broad-spectrum neutralizing antibodies. Therefore, both VP7 and VP8 are preferred antigenic targets for rotavirus subunit vaccines.

[0004] Current PoRV prevention and control measures include all-in and all-out management, environmental disinfection, electrolyte support therapy, antibiotics to prevent secondary infection, commercial vaccination against type A, and colostrum passive immunization. However, the high genetic diversity and frequent rearrangement of PoRV make these measures difficult to control virus transmission and antigenic drift in the long term. In contrast, vaccination is considered a core prevention and control strategy because of its high cost-effectiveness, ability to establish herd immunity, and long-lasting protection. Commercially available inactivated vaccines, live attenuated vaccines, and genetically engineered vaccines have limited cross-protection against multiple serogroups and cannot meet the prevention and control needs of rapidly mutating PoRV. There is an urgent need to develop a new generation of vaccines that cover major serotypes and are safe and broadly effective.

[0005] Bacterial flagellin is a promising immunoadjuvant due to its ability to simultaneously activate humoral and cellular immunity, overcome oral tolerance, significantly promote the secretion of mucosal proinflammatory cytokines, and possess a high degree of structural plasticity. However, achieving widespread clinical application still faces numerous technical challenges. First, flagellin itself is highly immunogenic, which may trigger excessive immune and inflammatory responses, potentially damaging the body. Furthermore, pre-existing anti-flagellin antibodies in the body may induce immune tolerance through neutralization, thereby reducing or even ineffective secondary immunization. Therefore, effectively balancing the immunogenicity and safety of flagellin is a major challenge in its practical application. Second, a common optimization approach is to reduce immunogenicity by truncating the hypervariable region of flagellin or inserting exogenous genes to enhance its adjuvant activity. However, these modifications may disrupt the native structure of flagellin, thereby weakening its activity as an immunoadjuvant. Therefore, although bacterial flagellin has broad application prospects as an immune adjuvant, in order to achieve its widespread clinical application and further optimization, several key technical difficulties such as immunogenicity, safety, structural stability and functional activity still need to be solved. 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 based on the dual targets of porcine rotavirus VP7 and VP8 and its preparation method and application. S.T It is a molecular skeleton and endogenous adjuvant. It significantly enhances immunogenicity by precisely embedding the dominant B cell neutralizing epitopes of the VP7 and VP8 proteins of PoRV in its hypervariable region and utilizing the self-adjuvant effect of the fusion protein.

[0007] Technical solution: In order to solve the above technical problems, the present invention provides a multi-epitope fusion protein, wherein the multi-epitope fusion protein is a Salmonella typhimurium flagellin FliC S.T The neutralizing epitopes of porcine rotavirus VP7 and VP8 proteins were replaced with the backbone protein FliC S.TThe neutralizing epitope of the VP7 protein has low immunogenicity and is exposed on the surface of the protein. The amino acid sequence of the neutralizing epitope of the VP8 protein is shown in SEQ ID NO.1-2, the amino acid sequence of the neutralizing epitope of the VP8 protein is shown in SEQ ID NO.5-6, and the amino acid sequence of the skeleton protein is shown in SEQ ID NO.11.

[0008] The nucleotide sequence of the nucleic acid molecule encoding the neutralizing epitope of the VP7 protein is shown in SEQ ID NO.3-4, the nucleotide sequence of the nucleic acid molecule encoding the neutralizing epitope of the VP8 protein is shown in SEQ ID NO.7-8, and the nucleotide sequence of the nucleic acid molecule encoding the backbone protein FliC is shown in SEQ ID NO. S.T The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.12.

[0009] Wherein, the amino acid sequence of the multi-epitope fusion protein is shown in SEQ ID NO.9.

[0010] The present invention also includes a nucleic acid molecule encoding the multi-epitope fusion protein. The DNA sequence of the nucleic acid molecule of the multi-epitope fusion protein is shown in SEQ ID NO.10.

[0011] The present invention also includes an expression cassette, a recombinant vector, a recombinant cell or a recombinant vector, including the nucleic acid molecule of the multi-epitope fusion protein.

[0012] The present invention also includes a method for preparing the multi-epitope fusion protein, comprising the following steps:

[0013] (1) respectively obtaining nucleic acid molecules encoding the neutralizing epitope of the VP7 protein and the neutralizing epitope of the VP8 protein, wherein the nucleotide sequence of the nucleic acid molecule encoding the neutralizing epitope of the VP7 protein is shown in SEQ ID NOs. 3 to 4, and the nucleotide sequence of the nucleic acid molecule encoding the neutralizing epitope of the VP8 protein is shown in SEQ ID NOs. 7 to 8;

[0014] (2) connecting the nucleic acid molecule described in step (1) with a plasmid to obtain a recombinant expression plasmid;

[0015] (3) The recombinant expression 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.

[0016] Wherein, the recombinant plasmid in step (1) is pUC57-FliC S.T -VP7+VP8 or pET-28a(+)-FliC S.T -VP7+VP8.

[0017] The present invention also includes the use of the multi-epitope fusion protein, the nucleic acid molecule of the multi-epitope fusion protein, the expression cassette, the recombinant vector, the recombinant cell or the recombinant vector 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 includes a vaccine or antibody, wherein the vaccine includes the multi-epitope fusion protein; the antibody is induced after immunizing an animal with the multi-epitope fusion protein.

[0020] The present invention also provides a FliC S.T -The method for constructing the VP7+VP8 multi-epitope fusion protein specifically comprises the following steps:

[0021] (1) Using the chimeric plasmid pUC57-FliC S.T -VP7+VP8 were used as DNA templates and PCR amplified with specific primers P1 and P2 to obtain the target gene fragment;

[0022] (2) The pET-28a(+) plasmid and purified FliC S.T -VP7+VP8 target gene fragments were double-digested, and the digested products were purified and ligated with T4 DNA ligase and transformed into TOP10 competent cells to obtain the recombinant plasmid pET-28a(+)-FliC S.T -VP7+VP8;

[0023] (3) The recombinant expression plasmid pET-28a(+)-FliC S.T -VP7+VP8 were transformed into Escherichia coli BL21 (DE3), and after induction of expression, the cells were broken by ultrasonic wave, and the inclusion bodies obtained by centrifugation were dissolved, renatured and purified to obtain the FliC S.T -VP7+VP8 multi-epitope fusion recombinant protein.

[0024] Furthermore, in step (1), the sequences of primers P1 and P2 used in the PCR amplification process are shown as SEQ ID NO.13 and SEQ ID NO.14.

[0025] Furthermore, in step (2), the specific system of the double enzyme digestion reaction is: 86 μL of the target gene fragment or the pET-28a(+) vector plasmid, 2 μL each of BamHI-HF and SacI-HF endonucleases, and 10 μL of 10×CutSmart buffer. After thorough mixing, the reaction is carried out in a constant temperature water bath at 37°C for 2.5 hours.

[0026] The present invention also provides a PoRV multi-epitope vaccine, wherein the PoRV multi-epitope vaccine comprises the FliC S.T -VP7+VP8 multi-epitope fusion protein.

[0027] The present invention also provides a FliC S.T -Application of VP7+VP8 multi-epitope fusion protein in the preparation of vaccines for the prevention of PoRV.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The present invention is based on the flagellin FliC of Salmonella typhimurium S.T The self-contained adjuvant function adopts the multi-epitope fusion antigen (MEFA) platform to precisely embed the advantageous neutralizing B cell epitopes of PoRV VP7 and VP8 into FliC S.T Hypervariable regions were used to construct a multi-antigen fusion protein with both broad neutralizing activity and high immunogenicity. This fusion protein demonstrated excellent neutralization and cellular immune responses in vitro and in mouse models, providing an innovative strategy for the efficient development of PoRV subunit vaccines. The advantages of this invention include the following:

[0029] (1) Self-adjuvant effect: fusion protein carries FliC S.T The adjuvant activity of the skeleton can significantly activate the host's innate and adaptive immunity without the need for additional adjuvants, increase the levels of neutralizing antibodies and T cell responses, thereby simplifying vaccine formulations and reducing production costs.

[0030] (2) Dual-target synergistic enhancement: The dual-target design of VP7 and VP8 can simultaneously induce potent neutralizing antibodies and cytotoxic T cell responses against PoRV, achieving synergistic protection of humoral immunity and cellular immunity.

[0031] In summary, based on the MEFA technology platform, the present invention combines the dominant B cell neutralizing epitopes of VP7 and VP8 of PoRV with FliC S.T By combining the autologous adjuvant skeleton, a multi-epitope fusion protein with both efficient neutralization and cellular immune activity was successfully constructed, providing new ideas and effective candidate antigens for the development of porcine rotavirus vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 For FliC S.T -Schematic diagram of the construction of VP7+VP8 multi-epitope fusion protein and its predicted structure. Figure 1 A is FliC S.T -Schematic diagram of the construction of VP7+VP8 multi-epitope fusion protein, Figure 1 B is FliC S.T -Prediction of the secondary structure of VP7+VP8 multi-epitope fusion protein; Figure 1C is FliC S.T -Prediction of the tertiary structure of VP7+VP8 multi-epitope fusion protein.

[0033] Figure 2 For FliC S.T -PCR amplification results of VP7+VP8 chimeric gene, where lane M is 2K PlusⅡDNAMarker; lane 1 is FliC S.T -VP7+VP8 chimeric gene; lane 2 is the negative control.

[0034] Figure 3 For the recombinant expression plasmid pET-28a(+)-FliC S.T - PCR identification electrophoresis diagram of VP7+VP8, where lane M is 2K PlusⅡ DNA Marker; lane 1 is the recombinant expression plasmid pET-28a(+)-FliC S.T -PCR amplification results of VP7+VP8; lane 2 is the negative control.

[0035] Figure 4 For purified FliC S.T - SDS-PAGE of VP7+VP8 multi-epitope fusion protein, where M is the protein molecular weight standard; lane 1 is the purified FliC S.T -VP7+VP8 multi-epitope fusion protein; Lane 2 is purified FliC S.T Recombinant protein; lane 3 is the purified VP7 recombinant protein, and lane 4 is the purified VP8 recombinant protein.

[0036] Figure 5 This is a western blot identification diagram of the purified VP7 and VP8 recombinant proteins. Figure 5 A is VP7 recombinant protein and anti-FliC S.T -VP7+VP8 immune serum antibody response graph; Figure 5 B is VP8 recombinant protein and anti-FliC S.T -VP7+VP8 immune serum antibody response diagram. Figure 6 ELISA for detecting FliC S.T -Specific anti-VP7 and anti-VP8 antibody levels in the serum of mice immunized with VP7+VP8 multi-epitope fusion protein. Figure 6 A is the anti-VP7 antibody titer; Figure 6 B is the anti-VP8 antibody titer.

[0037] Figure 7 ELISA was used to detect the concentrations of TNF-α and IL-6 in the supernatant of spleen cells from mice immunized with different reagents.

[0038] Figure 8For FliC S.T -Neutralizing antibody titer of serum from mice immunized with VP7+VP8 multi-epitope fusion protein against G5 and G9 PoRV viruses. DETAILED DESCRIPTION

[0039] Several exemplary embodiments of the present invention will be described in detail below. It should be clearly pointed out that the following description is intended to provide a more detailed description of certain aspects, features and embodiments of the present invention, and should not be considered as any limitation of the present invention.

[0040] It should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. In addition, for the numerical ranges involved in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is clearly disclosed. All intermediate values ​​within any given numerical value or numerical range, as well as any smaller ranges formed between these intermediate values, should also be considered to be included within the scope of the present invention. The upper and lower limits of the above-mentioned smaller ranges may be inclusive or exclusive, both of which fall within the scope of protection of the present invention.

[0041] Unless otherwise indicated, all technical and scientific terms used herein shall have the meanings commonly understood by those skilled in the art. Although preferred technical methods and experimental materials are described herein, any methods and materials that are substantially equivalent or functionally similar to those described herein may be used in actual applications and testing without departing from the basic concepts of the present invention.

[0042] All documents cited in this specification are incorporated herein by reference in their entirety to further disclose and support the methods and / or materials related to the documents. If there is a conflict between this specification and any cited document, the content of this specification shall prevail.

[0043] Without departing from the spirit and substance of the present invention, those skilled in the art will be able to make various modifications or variations to the specific embodiments of the present invention, and such modifications will be apparent to those skilled in the art. Therefore, other embodiments that can be derived from the contents of this specification should also be considered as part of the present invention. It should be understood that the description and examples provided herein are for illustrative purposes only.

[0044] In addition, the terms "include", "comprising", "having" and "containing" used in the present invention should be interpreted as open terms, indicating that the content is not limited to the components or characteristics listed therein.

[0045] Unless otherwise stated, all chemical reagents, biochemical reagents and materials used in the present invention can be obtained through commercial channels.

[0046] Example 1 FliC S.T -Construction, expression and identification of VP7+VP8 multi-epitope fusion protein

[0047] (1)FliC S.T -Construction and structure prediction of VP7+VP8 multi-epitope fusion protein

[0048] First, log in to the UniProt database (https: / / www.uniprot.org / ) to download the VP7 protein (accession number: P32546), VP8 protein (accession number: A0A1S5WJ26) of PoRV and the flagellin FliC of Salmonella typhimurium. S.T (Accession number: P06179) complete amino acid sequence. Subsequently, the IEDB B cell epitope online prediction tool (http: / / tools.immuneepitope.org / bcell) was used to predict the linear B cell epitopes of the above three proteins. The specific prediction parameters are: setting the threshold of epitope probability to 0.5; selecting at least seven consecutive amino acid residues with an epitope probability score exceeding 0.5 as a putative B cell epitope. 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 screened dominant B cell neutralizing epitopes in VP7 protein (the amino acid and nucleotide sequences of the screened dominant B cell neutralizing epitopes VP7-E1 and VP7-E2 in VP7 protein are listed in SEQ ID NO. 1-2 and SEQ ID NO. 3-4, respectively) and the dominant B cell neutralizing epitopes in VP8 protein (the amino acid and nucleotide sequences of the dominant B cell neutralizing epitopes VP8-E1 and VP8-E2 in VP8 protein are listed in SEQ ID NO. 5-6 and SEQ ID NO. 7-8, respectively) were substituted for FliC. S.T The original epitopes with low immunogenicity and surface exposure on the backbone were used to construct FliC S.T -VP7+VP8 multi-epitope fusion protein (corresponding amino acid and nucleotide sequences are shown in SEQ ID NO.9 and SEQ ID NO.10), the specific construction diagram is as follows Figure 1 As shown in A.

[0049] Then, 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 model with the highest score was selected as the final prediction model. The three-dimensional structure of the model was analyzed using PyMOL software. The results showed that the insertion of exogenous VP7 and VP8 epitopes did not disrupt the scaffold protein FliC. S.TThe natural conformation of the protein; and the inserted epitopes are all located on the protein surface ( Figure 1 B. Figure 1 C).

[0050] (2)FliC S.T -PCR amplification of VP7+VP8, VP7 and VP8 genes

[0051] The plasmid pUC57-FliC was synthesized by Nanjing Qingke Biotechnology Co., Ltd. S.T -VP7+VP8, pUC57-VP7 and pUC57-VP8 (the insertion sites of the three genes are between the BamHI and SphI restriction sites of the pUC57 plasmid) were used as templates, and primers P1 (SEQ ID NO.13) and P2 (SEQ ID NO.14) were used to amplify FliC S.T -VP7+VP8 genes; primers P3 (SEQ ID NO.15) and P4 (SEQ ID NO.16) amplify VP7 gene; primers P5 (SEQ ID NO.17) and P6 (SEQ ID NO.18) amplify VP8 gene. The PCR reaction system was prepared as follows: pUC57-FliC S.T 4 μL of VP7+VP8 / pUC57-VP7 / pUC57-VP8 DNA template, 10 μL of 5× Pfu Buffer, 1 μL of Pfu enzyme (Beijing Quanshijin Biotechnology, Cat. No. AP221-01), 4 μL of 2.5 mM dNTP mix, 26 μL of ddH2O, and 2.5 μL each of primers P1 / P2, P3 / P4, and P5 / P6. The VP7 gene GenBank accession number is MH137265.1, and the VP8 gene GenBank accession number is JQ011467.1.

[0052] The PCR reaction conditions were set as follows: pre-denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 94°C for 40 sec, annealing at 52°C for 30 sec, extension at 72°C for 2 min, and finally extension at 72°C for 10 min and storage at 4°C for termination.

[0053] The amplified products were detected by 1.0% agarose gel electrophoresis. Figure 2 As shown, a single clear band appeared at about 1500 bp, which was consistent with FliC S.T -The expected fragment size of VP7+VP8 genes is consistent. The target band was then recovered using the DNA gel recovery kit (Cat. No. DP214-03) from Tiangen Biochemical Technology Co., Ltd. (3) pET-28a(+)-FliC S.T Construction of VP7+VP8, pET-28a(+)-VP7 and pET-28a(+)-VP8 recombinant plasmids

[0054] First, the purified FliC S.T -VP7+VP8 target gene fragments were double-enzyme digested with BamHI-HF (NEB, Catalog No.: R3136M) and SacI-HF (NEB, Catalog No.: R3156M); the purified VP7 gene fragments were double-enzyme digested with NheI-HF (NEB, Catalog No.: R3131M) and SalI-HF (NEB, Catalog No.: R3138M); the purified VP8 gene fragments were double-enzyme digested with BamHI-HF (NEB, Catalog No.: R3136M) and SalI-HF (NEB, Catalog No.:

[0055] R3138M) for double digestion; pET-28a(+) plasmid was double digested with the corresponding endonucleases. The digestion system (100 μL) included: 86 μL of the target gene fragment or pET-28a(+) vector, 2 μL of each endonuclease, and 10 μL of 10× CutSmart buffer. After the digestion reaction, the products were recovered and purified by agarose gel electrophoresis.

[0056] Purified FliC S.T -VP7+VP8, VP7 or VP8 fragments were ligated with linearized pET-28a(+) vector using T4 DNA ligase (NEB, 10 U / μL) at 16°C overnight. The total volume of the ligation reaction was 10 μL, and the components were as follows: 4 μL of pET-28a(+) linearized vector, target gene fragment FliC S.T -VP7+VP8, VP7 or VP8 4 μL, 10×T4 DNA ligase buffer 1 μL and T4 DNA ligase 1 μL.

[0057] The ligation product was then transformed into E. coli TOP10 competent cells. After transformation, 1 mL of resistance-free LB liquid medium was added and the cells were shaken at 37°C, 220 rpm for 2 hours for recovery. The bacterial suspension was spread onto LB plates containing 50 μg / mL kanamycin and incubated inverted at 37°C overnight. The next day, multiple single clones were selected from the plates and cultured overnight in 5 mL of LB liquid medium containing the same concentration of kanamycin.

[0058] Using overnight culture solution as template, preliminary identification was performed by PCR. Figure 3 As shown, the suspected positive clone showed a single band at about 1500bp, which was consistent with the expected size of the target fragment. The PCR-positive samples were sent to the company for DNA sequencing, and the results confirmed that they were correct. Finally, the recombinant plasmid pET-28a(+)-FliC was obtained. S.T-VP7+VP8, pET-28a(+)-VP7 and pET-28a(+)-VP8.

[0059] (4)pET-28a(+)-FliC S.T -Expression and identification of VP7+VP8, VP7 and VP8 recombinant proteins

[0060] The recombinant expression plasmid was transformed into the E. coli expression host strain BL21 (DE3). A single positive clone was picked and inoculated into 5 mL LB liquid medium containing 30 μg / mL kanamycin sulfate. The culture was shaken at 37°C and 220 rpm for 16 hours. S.T -VP7+VP8 / BL21 recombinant expression bacteria were identified by PCR using primers P1 (SEQ ID NO.13) and P2 (SEQ ID NO.14), and pET-28a(+)-VP7 / BL21 recombinant expression bacteria were identified by PCR using primers P3 (SEQ ID NO.15) and P4 (SEQ ID NO.16); pET-28a(+)-VP8 / BL21 recombinant expression bacteria were identified by PCR using primers P5 (SEQ ID NO.17) and P6 (SEQ ID NO.18) to confirm correctness.

[0061] Subsequently, the overnight culture was inoculated into 500 mL of LB medium containing 30 μg / mL kanamycin sulfate at a ratio of 1:100 and cultured in a shaking incubator at 37°C until OD 600 When the protein level reached 0.6-0.8, IPTG was added at a final concentration of 1 mmol / L and the induction culture was continued for 4 hours. The induced bacteria were collected, ultrasonically disrupted and centrifuged, the precipitate was recovered and fully dissolved with inclusion body solution. After centrifugation again, the supernatant was taken and the recombinant protein was purified using nickel ion affinity chromatography column (Ni-NTA). The purified product was analyzed by SDS-PAGE electrophoresis and showed a clear protein band at about 55 kDa, which was similar to FliC. S.T -The theoretical molecular weight of VP7+VP8 multi-epitope fusion protein is consistent ( Figure 4 The protein concentration was determined by BCA method. S.T -The concentration of VP7+VP8 recombinant protein was 1.09 mg / mL, the concentration of VP7 recombinant protein was 0.68 mg / mL, and the concentration of VP8 recombinant protein was 0.92 mg / mL.

[0062] Example 2FliC S.T -Analysis of immunogenicity of VP7+VP8 multi-epitope fusion protein

[0063] (1) Mouse immunization

[0064] Twenty-five 7-week-old female BALB / c mice were randomly divided into five groups, with five mice in each group. The first group of mice was subcutaneously injected with 50 μg of FliC S.T -VP7+VP8 recombinant protein; the second group of mice were subcutaneously injected with 50 μg VP7 recombinant protein (emulsified with an equal volume of complete Freund's adjuvant); the third group of mice were subcutaneously injected with 50 μg VP8 recombinant protein (emulsified with an equal volume of complete Freund's adjuvant); the fourth group of mice were subcutaneously injected with 50 μg purified FliC S.T Recombinant protein (Pang et al., 2024); the fifth 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; incomplete Freund's adjuvant was used for the second and third immunizations in groups 2 and 3. Blood was collected from the retroorbital vein before immunization 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.

[0065] (2) Western blot identification experiment

[0066] To further verify the function of the antigenic epitope of the fusion protein, its immunoreactivity was identified by western blot. The purified VP7 or VP8 recombinant protein samples were loaded onto 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. Afterwards, the PVDF membrane was incubated with a 1:6000 dilution of FliC S.T Incubate the membrane with serum from mice immunized with VP7 and VP8 recombinant proteins at 4°C for 1.5 hours. After washing with PBST, add HRP-conjugated goat anti-mouse IgG (ABclonal, Catalog No. AS003) at a dilution of 1:10,000 and incubate for another 1.5 hours at room temperature. After multiple washes with PBST, develop the membrane using ECL. After a 90-second reaction in the dark, the antigen-antibody binding signal was detected using a chemiluminescence imaging system.

[0067] The results are as follows Figure 5 As shown, the purified VP7 and VP8 recombinant proteins could be cleaved by FliC S.T -VP7+VP8 recombinant protein was specifically recognized by immune serum, indicating that FliC S.T -VP7+VP8 fusion protein retains the immunogenicity of both VP7 and VP8 inserted epitopes and has good antigen epitope display function.

[0068] (3) Detection of anti-VP7 and anti-VP8 specific IgG antibodies

[0069] Purified VP7 or VP8 recombinant protein was diluted in ELISA coating buffer (0.05 M carbonate buffer, pH 9.6) and added to a 96-well plate at 500 μg / well. Incubate at 37°C for 1 hour and then transfer to a 4°C refrigerator overnight. The next day, equilibrate at room temperature for 30 minutes, wash the plate three times with PBST (0.05% Tween-20) (300 μL / well, 5 minutes each time), add 200 μL / well of blocking buffer (10% skim milk powder / PBST), and incubate at 37°C for 1 hour. Wash the plate as above. The mouse immune serum was diluted gradiently with PBST (1:200-1:12800, specifically 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800), with 3 replicate wells for each dilution, 100 μL / well, and incubated at 37°C for 90 min. Wash the plate 3 times. Add 100 μL / well HRP-labeled goat anti-mouse IgG (secondary antibody, 1:5000 / PBST) and incubate at 37°C in the dark for 90 min. Wash the plate three more times with PBST. Add 200 μL TMB substrate to each well, develop color at 37°C in the dark for 30 min, terminate the reaction, and measure the absorbance (OD) at 650 nm. The titer of anti-VP7 and VP8 antibodies is determined as follows: according to the OD of the serum sample 650n m 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 -VP7+VP8 multi-epitope fusion protein immunization group can simultaneously induce high titer anti-VP7 and anti-VP8 specific IgG, among which the final antibody titer (log 10 ) were 3.28±0.13 and 3.31 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 -VP7+VP8 multi-epitope fusion protein has good immunogenicity.

[0070] (4) Detection of cytokines in spleen cell culture supernatant

[0071] On the 14th day after the last immunization, the mice were killed by painless cervical dislocation. The spleen was removed under sterile conditions and placed in a sterile culture dish containing RPMI 1640 culture medium. It was gently ground with a syringe piston with a needle until the tissue was completely dissociated. After washing the cell suspension with RPMI 1640 three times, the cell pellet was collected by centrifugation at 1000r / min for 10 minutes. 1× red blood cell lysis buffer (Beijing Solebow Technology Co., Ltd., catalog number: R1010) was added, gently mixed and allowed to stand at room temperature for 5 minutes until the red blood cells were completely lysed, and then centrifuged again at 1000r / min for 10 minutes to discard the supernatant. Resuspend the cells with sterile PBS and repeat the centrifugation to completely remove the lysis buffer residue, and then resuspend with RPMI containing 0.5% FBS and 1% penicillin-streptomycin-double antibody.

[0072] Resuspend the cells in 1640 medium and adjust the cell density to 2.5 × 10 5 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 FliC was added to each well at a final concentration of 5 μg / mL. S.T -VP7+VP8 recombinant proteins were placed in a 37°C, 5% CO2 incubator for 72 hours. After the culture, the supernatant was collected and the concentrations of IFN-γ (Shenzhen Xinbosheng Biotechnology Co., Ltd., catalog number EMC101g) and IL-6 (Shenzhen Xinbosheng Biotechnology Co., Ltd., catalog number EMC004) were measured according to the kit instructions. The results are as follows. Figure 7 As shown in the results, the levels of IFN-γ and IL-6 in the supernatant of spleen cells from mice immunized with the multi-epitope fusion protein 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 good activation efficacy at the cellular immunity level.

[0073] (5) Neutralizing antibody detection

[0074] In a 96-well plate, 100 μL of serum-free DMEM medium (Gibco, Cat. No. 6125197) containing 10 μg / mL trypsin was added to each well. 100 μL of the immune serum from the five groups of mice prepared in step (1) of Example 2 was added to the first well, and after thorough mixing, it was serially diluted in a 1:2 ratio to a 1:2 ratio. 11 4 replicate wells were set up for each dilution. Then, 100 μL of 200 TCID 50The G5 and G9 PoRV virus solutions (preserved in the laboratory) were 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 To evaluate the virus titer, 100 TCID 50 The virus solution was diluted 10-fold (10 0 ~10 -3 ) with 100 μL per well, with a total of 6 replicate wells, to conduct a virus regression test. All wells were incubated at 37°C for 48 h.

[0075] After the culture is completed, the culture medium is discarded and the plate is washed 3 times with PBS; 150 μL of 80% acetone pre-cooled to -20°C is added to each well and fixed at 4°C for 30 minutes; the plate is washed again with PBS 3 times; it is blocked with 5% skim milk / PBST at 37°C for 1 hour, and the plate is washed with PBST. Subsequently, 200-fold diluted immune sera of 5 groups of mice prepared in step (1) of Example 2 are added to each well after inactivation (100 μL / well), and incubated at 37°C in the dark for 1 hour; after washing the plate with PBST, a 500-fold diluted fluorescent-labeled secondary antibody (100 μL / well) is added, and incubated at 37°C in the dark for 1 hour; the plate is washed 3 times with PBST. Finally, the number of infected cells in each well is observed and recorded by fluorescence microscopy, and the neutralizing antibody titer is calculated based on this. The results are as follows Figure 8 As shown, FliC S.T -The neutralization titers of the serum from mice immunized with VP7+VP8 multi-epitope fusion protein against G5 and G9 PoRV virus solutions were 1:2 9 and 1:2 10 , verifying its good immunogenicity and showing its potential as a candidate epitope vaccine for PoRV.

Claims

1. A multi-epitope fusion protein, characterized in that: The multi-epitope fusion protein is based on the flagellin FliC of Salmonella typhimurium S.T The neutralizing epitopes of porcine rotavirus VP7 and VP8 proteins were replaced with the backbone protein FliC S.T The neutralizing epitope of the VP7 protein is constructed by targeting a B cell epitope with low immunogenicity and exposed on the surface of the protein. The amino acid sequence of the neutralizing epitope of the VP7 protein is shown in SEQ ID NO. 1 to 2, the amino acid sequence of the neutralizing epitope of the VP8 protein is shown in SEQ ID NO. 5 to 6, and the backbone protein FliC S.T The amino acid sequence is shown in SEQ ID NO.

11.

2. The multi-epitope fusion protein according to claim 1, characterized in that The nucleotide sequence of the nucleic acid molecule encoding the neutralizing epitope of the VP7 protein is shown in SEQ ID NO.3-4, the nucleotide sequence of the nucleic acid molecule encoding the neutralizing epitope of the VP8 protein is shown in SEQ ID NO.7-8, and the nucleotide sequence of the nucleic acid molecule encoding the backbone protein FliC is shown in SEQ ID NO. S.T The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

12.

3. The multi-epitope fusion protein according to claim 1, characterized in that The amino acid sequence of the multi-epitope fusion protein is shown in SEQ ID NO.

9.

4. A nucleic acid molecule encoding the multi-epitope fusion protein according to any one of claims 1 to 3, characterized in that: The DNA sequence of the nucleic acid molecule of the multi-epitope fusion protein is shown in SEQ ID NO.

10.

5. An expression cassette, a recombinant vector, a recombinant cell or a recombinant vector, characterized in that: A nucleic acid molecule comprising the multi-epitope fusion protein according to claim 4.

6. The method for preparing the multi-epitope fusion protein according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) respectively obtaining nucleic acid molecules encoding the neutralizing epitope of the VP7 protein and the neutralizing epitope of the VP8 protein, wherein the nucleotide sequence of the nucleic acid molecule encoding the neutralizing epitope of the VP7 protein is shown in SEQ ID NOs. 3 to 4, and the nucleotide sequence of the nucleic acid molecule encoding the neutralizing epitope of the VP8 protein is shown in SEQ ID NOs. 7 to 8; (2) connecting the nucleic acid molecule described in step (1) with a plasmid to obtain a recombinant expression plasmid; (3) The recombinant expression 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.

7. The method for preparing a multi-epitope fusion protein according to claim 6, wherein: The recombinant plasmid in step (1) is pUC57-FliC S.T -VP7+VP8 or pET-28a(+)-FliC S.T -VP7+VP8.

8. Use of the multi-epitope fusion protein according to any one of claims 1 to 3, the nucleic acid molecule of the multi-epitope fusion protein according to claim 4, the expression cassette, recombinant vector, recombinant cell or recombinant vector according to claim 5 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 1 to 3; the antibody comprises the antibody induced by immunizing an animal with the multi-epitope fusion protein according to any one of claims 1 to 3.

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