Porcine rotavirus vp7 and vp8 double-target based polyepitope fusion protein and preparation method and application thereof

Abstract

The application discloses a multi-epitope fusion protein based on porcine rotavirus VP7 and VP8 double targets and a preparation method and application thereof. S.T As a skeleton, the advantages of precisely integrating VP7 and VP8 neutralizing epitopes are constructed into multi-epitope fusion antigen FliC S.T -VP7+VP8. The fusion protein has two immunological advantages: FliC S.T The skeleton has TLR5 adjuvant activity, can efficiently activate the innate immunity and adaptive immunity of a host, and can synchronously present key neutralizing epitopes of porcine rotavirus VP7 and VP8 through a multi-epitope strategy, so that high-titer specific neutralizing antibodies and strong cellular immune responses against the two structural proteins can be induced in the body. The application provides a multi-epitope fusion antigen of porcine rotavirus which integrates high immunogenicity and broad-spectrum neutralization protection, and provides a feasible technical scheme for the prevention and control of porcine rotavirus.

Description

Technical Field

[0001] This invention relates to the field of vaccine biotechnology, specifically to a multi-epitope fusion protein based on porcine rotavirus VP7 and VP8 dual targets, its preparation method, and its application. Background Technology

[0002] Porcine rotavirus (PoRV) is one of the major pathogens facing the global swine industry, posing a particularly serious threat to suckling piglets. Epidemiological surveys show that 40%–60% of pig herds test positive for PoRV, with more than 10% of piglets exhibiting related clinical symptoms. The severity of PoRV is significantly amplified, especially when co-infected with pathogens such as porcine epidemic diarrhea virus (PEDV) or enterotoxigenic Escherichia coli (ETEC). PoRV primarily attacks the mature epithelial cells of the small intestinal villi in piglets, causing villus atrophy and crypt hyperplasia, leading to malabsorption and diarrhea lasting 3–4 days. Although the direct mortality rate caused by this virus is relatively low, it significantly delays piglet growth, reduces feed utilization, and affects weaning weight and survival rate. Five serogroups of PoRV exist: A, B, C, E, and H, with serogroup A being the most prevalent. It is worth noting that the detection rate of type C PoRV has risen rapidly in recent years; in my country, the infection rate in piglets under 3 days old has exceeded 30%, indicating that type C PoRV has become an important pathogen of enteritis in newborn piglets. This virus is highly stable in the environment and requires effective inactivation using chlorine-containing disinfectants. Diagnosis mainly relies on reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), and it is necessary to rule out mixed infections 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 non-structural proteins (NSP1–NSP6). VP7 and VP4 together form the viral smooth coat, determining the G type and inducing neutralizing antibody production. VP4, after trypsin cleavage, produces the VP8 subunit, which binds to sialic acid or HBGAs on the host cell surface, mediating viral adsorption. Compared to VP5, VP8 exhibits higher immunogenicity, better stability, and higher expression yield. Furthermore, its antibodies can not only neutralize homologous viruses but also provide cross-protection against different P types. 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 control measures include all-in, all-out management, environmental disinfection, electrolyte support therapy, antibiotics to prevent secondary infections, commercial vaccination against type A, and passive immunization with colostrum. However, the high genetic diversity and frequent rearrangements of PoRV make these measures insufficient for long-term control of viral transmission and antigenic drift. In contrast, vaccination is considered a core control strategy due to its cost-effectiveness, ability to establish herd immunity, and durable protection. Commercially available inactivated vaccines, live attenuated vaccines, and genetically engineered vaccines offer limited cross-protection against multiple serotypes, failing to meet the needs of controlling rapidly mutating PoRV. There is an urgent need to develop next-generation vaccines that cover major serotypes and possess both safety and broad-spectrum efficacy.

[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 pro-inflammatory factors in the mucosa, and possess high structural plasticity. However, its widespread clinical application faces numerous technical challenges. First, flagellin itself has strong immunogenicity, which may trigger excessive immune and inflammatory responses, potentially causing harm to the body. Simultaneously, existing anti-flagellate antibodies may induce immune tolerance through neutralization, leading to reduced or ineffective re-immunization. Therefore, effectively balancing the immunogenicity and safety of flagellin is a major challenge in its practical application. Second, common optimization methods involve truncating the hypervariable regions of flagellin or inserting exogenous genes to reduce immunogenicity and enhance its adjuvant effect. 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, several key technical challenges, such as immunogenicity, safety, structural stability, and functional activity, still need to be addressed to achieve its widespread clinical application and further optimization. Summary of the Invention

[0006] Objective of the Invention: The technical problem to be solved by this invention is to provide a multi-epitope fusion protein based on porcine rotavirus VP7 and VP8 dual targets, its preparation method, and its applications. This fusion protein innovatively utilizes the flagellated protein FliC from Salmonella Typhimurium. S.T As a molecular backbone and endogenous adjuvant, it significantly enhances immunogenicity by precisely embedding the VP7 and VP8 proteins of PoRV into their hypervariable regions in dominant B cells and epitopes, utilizing the self-adjuvant effect of the fusion protein.

[0007] Technical Solution: To solve the above-mentioned technical problems, the present invention provides a multi-epitope fusion protein, wherein the multi-epitope fusion protein is based on the Salmonella Typhimurium flagellate protein FliC. S.T As the backbone, the neutralizing epitopes of porcine rotavirus VP7 and VP8 proteins were replaced with the backbone protein FliC. S.TThe protein was constructed from B cell epitopes with low immunogenicity and exposed on the protein surface. The amino acid sequences of the neutralizing epitopes of the VP7 protein are shown in SEQ ID NO. 1-2, the amino acid sequences of the neutralizing epitopes of the VP8 protein are shown in SEQ ID NO. 5-6, and the amino acid sequences of the cytoskeletal protein are shown in SEQ ID NO. 11.

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

[0009] 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 which is shown in SEQ ID NO.10.

[0011] The present invention also includes expression cassettes, recombinant vectors, recombinant cells or recombinant vectors, including nucleic acid molecules of the multi-epitope fusion protein.

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

[0013] (1) Obtain nucleic acid molecules encoding neutralizing epitopes of VP7 protein and VP8 protein respectively. The nucleotide sequences of the nucleic acid molecules encoding the neutralizing epitopes of VP7 protein are shown in SEQ ID NO.3-4, and the nucleotide sequences of the nucleic acid molecules encoding the neutralizing epitopes of VP8 protein are shown in SEQ ID NO.7-8.

[0014] (2) The nucleic acid molecules described in step (1) are linked to the plasmid to obtain a recombinant expression plasmid;

[0015] (3) The recombinant expression plasmid was transferred into E. coli culture, induced to express, and then broken by ultrasonication, centrifuged and purified to obtain the multi-epitope fusion protein.

[0016] The recombinant plasmid mentioned 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 application 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] The drugs mentioned include vaccines, antibodies, or diagnostic reagents.

[0019] The present invention also includes a vaccine or antibody, wherein the vaccine comprises the aforementioned multi-epitope fusion protein; and the antibody comprises being induced to be produced after immunizing an animal with the multi-epitope fusion protein.

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

[0021] (1) The chimeric plasmid pUC57-FliC S.T Using VP7+VP8 as a DNA template, PCR amplification was performed with specific primers P1 and P2 to obtain the target gene fragment.

[0022] (2) pET-28a(+) plasmid and purified FliC S.T The target gene fragment (VP7+VP8) was subjected to double enzyme digestion. After purification, the digested products were 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 was transformed into E. coli BL21(DE3), and after induction of expression, the bacterial cells were disrupted by sonication. The inclusion bodies obtained by centrifugation were dissolved, refolded, 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 in SEQ ID NO.13 and SEQ ID NO.14.

[0025] Furthermore, in step (2), the specific system for the double enzyme digestion reaction is as follows: 86 μL of the target gene fragment or the pET-28a(+) vector plasmid, 2 μL each of BamHI-HF and SacI-HF restriction enzymes, 10 μL of 10×CutSmart buffer, and 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 multiepitope vaccine, wherein the PoRV multiepitope vaccine comprises FliC as described above. S.T -VP7+VP8 multi-epitope fusion protein.

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

[0028] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The present invention is based on the flagellated protein FliC of Salmonella Typhimurium. S.T With its inherent adjuvant function, FliC utilizes the multi-epitope fusion antigen (MEFA) platform to precisely embed the beneficial neutralizing B-cell epitopes of PoRV VP7 and VP8 into the FliC platform. S.T The hypervariable region was used to construct a multi-antigen fusion protein with both broad-spectrum neutralizing activity and high immunogenicity. This fusion protein exhibited excellent neutralizing capacity and cellular immune response in both in vitro and mouse models, providing an innovative strategy for the efficient development of PoRV subunit vaccines. The advantages of this invention specifically include the following aspects:

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

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

[0031] In summary, based on the MEFA technology platform, this invention integrates the VP7 and VP8 dominant B cell neutralization epitopes of PoRV with FliC. S.T By combining autologous adjuvant backbone, a multi-epitope fusion protein with both high neutralizing and cellular immune activity was successfully constructed, providing a new approach and effective candidate antigen for the development of porcine rotavirus vaccines. Attached Figure Description

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

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

[0034] Figure 3 The recombinant expression plasmid pET-28a(+)-FliC S.T Electrophoresis image of PCR identification of VP7+VP8, where lane M is the 2K Plus II 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 image of the -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 contains purified FliC. S.T Recombinant proteins; lane 3 contains purified VP7 recombinant protein, and lane 4 contains purified VP8 recombinant protein.

[0036] Figure 5 Western blot illustration of purified VP7 and VP8 recombinant proteins. Figure 5 A represents VP7 recombinant protein and anti-FliC. S.T -VP7+VP8 immune serum antibody reaction diagram; Figure 5 B represents VP8 recombinant protein and anti-FliC. S.T -VP7+VP8 immune serum antibody reaction diagram. Figure 6 For ELISA detection of FliC S.T - VP7+VP8 multi-epitope fusion protein immunized mice serum specific anti-VP7 and anti-VP8 antibody levels. Figure 6 A represents the titer of anti-VP7 antibody; Figure 6 B represents the titer of anti-VP8 antibody.

[0037] Figure 7 The concentrations of TNF-α and IL-6 in the culture supernatant of spleen cells from mice immunized with different reagents were detected by ELISA.

[0038] Figure 8For FliC S.T -VP7+VP8 multi-epitope fusion protein-immunized mouse serum neutralizing antibody titer against G5 and G9 PoRV viruses. Detailed Implementation

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

[0040] It should be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of protection of this invention. Furthermore, the numerical ranges involved in this invention should be understood to explicitly disclose every intermediate value between the upper and lower limits of the range. All intermediate values ​​within any given numerical value or range, as well as any smaller range formed between these intermediate values, should also be considered to be included within the scope of this invention. The upper and lower limits of the aforementioned smaller ranges can be inclusive or exclusionary, both of which fall within the scope of protection of this invention.

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

[0042] All references cited in this specification are incorporated herein by reference in their entirety to further disclose and support the methods and / or materials associated with those references. In the event of any conflict between this specification and any cited reference, the contents of this specification shall prevail.

[0043] Without departing from the spirit and essence of this invention, those skilled in the art will be able to make various improvements or modifications to the specific embodiments of this invention, and such modifications will be obvious to those skilled in the art. Therefore, other embodiments that can be derived from the content of this specification should also be considered part of this invention. It should be understood that the descriptions and embodiments provided herein are for illustrative purposes only.

[0044] Furthermore, the terms “comprising,” “including,” “having,” and “containing” used in this invention should be interpreted as open-ended terms, indicating that the content is not limited to the listed components or features.

[0045] Unless otherwise stated, all chemical reagents, biochemical reagents and materials used in this invention are commercially available.

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

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

[0048] First, log in to the UniProt database (https: / / www.uniprot.org / ) and download the PoRV VP7 protein (accession number: P32546), VP8 protein (accession number: A0A1S5WJ26), and Salmonella typhimurium flagellate protein FliC. S.T The complete amino acid sequence of (accession number: P06179) was then used. Subsequently, the IEDB B-cell epitope online prediction tool (http: / / tools.immuneepitope.org / bcell) was used to predict linear B-cell epitopes of the above three proteins. Specific prediction parameters were: an epitope probability threshold of 0.5 was set; epitopes with at least seven consecutive amino acid residues having a probability score exceeding 0.5 were selected as a putative B-cell epitope. Based on this, relying on the MEFA technology platform, FliC... S.T As a backbone protein (whose amino acid and nucleotide sequences are listed in SEQ ID NO. 11 and SEQ ID NO. 12, respectively), the selected dominant B cell neutralization epitopes in the VP7 protein (the amino acid and nucleotide sequences of the selected dominant B cell neutralization epitopes VP7-E1 and VP7-E2 in the VP7 protein are listed in SEQ ID NO. 1-2 and SEQ ID NO. 3-4, respectively) and the dominant B cell neutralization epitopes in the VP8 protein (the amino acid and nucleotide sequences of the dominant B cell neutralization epitopes VP8-E1 and VP8-E2 in the VP8 protein are listed in SEQ ID NO. 5-6 and SEQ ID NO. 7-8, respectively) replaced FliC. S.T FliC was constructed using pre-existing epitopes with low immunogenicity on the backbone and exposed on the surface. 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 Figure A.

[0049] Next, homology modeling of the fusion protein sequence was performed using the Phyre2 platform. Based on key indicators such as model confidence, sequence coverage, and homology (>90%), the generated structural models were comprehensively scored, and the highest-scoring model was selected as the final prediction model. Three-dimensional structural analysis of the model was performed using PyMOL software. The results showed that the insertion of exogenous VP7 and VP8 epitopes did not disrupt the cytoskeleton protein FliC. S.TThe natural conformation; 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 Using VP7+VP8, pUC57-VP7, and pUC57-VP8 (the insertion sites of these three genes are all between the BamHI and SphI restriction sites of the pUC57 plasmid) as templates, FliC was amplified using primers P1 (SEQ ID NO.13) and P2 (SEQ ID NO.14), respectively. S.T -VP7+VP8 gene; primers P3 (SEQ ID NO.15) and P4 (SEQ ID NO.16) amplify the VP7 gene; primers P5 (SEQ ID NO.17) and P6 (SEQ ID NO.18) amplify the VP8 gene. The PCR reaction system is 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 TransGen Biotech, catalog number: AP221-01), 4 μL of 2.5 mM dNTP mixture, 26 μL of ddH2O, and 2.5 μL each of primers P1 / P2, P3 / P4, and P5 / P6. The VP7 gene has GenBank accession number MH137265.1, and the VP8 gene has GenBank accession number JQ011467.1.

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

[0053] The amplification products were detected by 1.0% agarose gel electrophoresis, and the results are as follows: Figure 2 As shown, a single clear band appears at approximately 1500 bp, consistent with FliC. S.T -VP7+VP8 gene fragments were of the same size. The target band was then recovered using a DNA gel recovery kit (catalog number DP214-03) from Tiangen Biotech Co., Ltd. (3) pET-28a(+)-FliC S.T Construction of recombinant plasmids pET-28a(+)-VP7 and pET-28a(+)-VP8

[0054] First, the purified FliC S.T The VP7 and VP8 target gene fragments were double-digested with BamHI-HF (NEB, catalog number: R3136M) and SacI-HF (NEB, catalog number: R3156M), respectively; the purified VP7 gene fragment was double-digested with NheI-HF (NEB, catalog number: R3131M) and SalI-HF (NEB, catalog number: R3138M), respectively; the purified VP8 gene fragment was double-digested with BamHI-HF (NEB, catalog number: R3136M) and SalI-HF (NEB, catalog number: R3138M), respectively.

[0055] The R3138M vector was double-digested; the pET-28a(+) plasmid was double-digested with the corresponding restriction enzyme. The digestion system was 100 μL, specifically including: 86 μL of the target gene fragment or pET-28a(+) vector, 2 μL of each restriction enzyme, 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 The VP7+VP8, VP7, or VP8 fragments were ligated overnight at 16°C with the linearized pET-28a(+) vector and T4 DNA ligase (NEB, 10 U / μL). The total ligation volume was 10 μL, and the components were as follows: 4 μL of the pET-28a(+) linearized vector and the target gene fragment FliC. S.T -VP7+VP8, VP7 or VP8 4μL, 10×T4 DNA ligase buffer 1μL and T4 DNA ligase 1μL.

[0057] The ligation product was then transformed into E. coli TOP10 competent cells. After transformation, 1 mL of antibiotic-free LB liquid medium was added, and the cells were incubated at 37°C with shaking at 220 rpm for 2 h. The bacterial culture was then spread onto LB agar plates containing 50 μg / mL kanamycin and incubated upside down at 37°C overnight. The next day, multiple single clones were picked from the plates and cultured overnight in 5 mL of LB liquid medium containing the same concentration of kanamycin.

[0058] Preliminary identification was performed by PCR using overnight bacterial culture as a template. Electrophoresis results are shown below. Figure 3 As shown, the suspected positive clone exhibited a single band at approximately 1500 bp, consistent with the expected size of the target fragment. Samples identified by PCR were sent to the company for DNA sequencing, and the results confirmed the correctness, ultimately yielding the recombinant plasmid pET-28a(+)-FliC. S.T-VP7+VP8, pET-28a(+)-VP7 and pET-28a(+)-VP8.

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

[0060] The successfully constructed 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 of LB broth containing 30 μg / mL kanamycin sulfate, and cultured at 37°C and 220 rpm for 16 h with shaking. Subsequently, pET-28a(+)-FliC... S.T The recombinant expression strain pET-28a(+)-VP7 / BL21 was identified by PCR using primers P1 (SEQ ID NO. 13) and P2 (SEQ ID NO. 14), the recombinant expression strain pET-28a(+)-VP7 / BL21 was identified by PCR using primers P3 (SEQ ID NO. 15) and P4 (SEQ ID NO. 16), and the recombinant expression strain pET-28a(+)-VP8 / BL21 was identified by PCR using primers P5 (SEQ ID NO. 17) and P6 (SEQ ID NO. 18), and the results were confirmed to be correct.

[0061] Subsequently, the overnight culture medium was inoculated at a ratio of 1:100 into 500 mL of LB medium containing 30 μg / mL kanamycin sulfate, and cultured at 37°C in a shaker until OD. 600 When the bacterial concentration reached 0.6–0.8, IPTG was added to a final concentration of 1 mmol / L and the culture was continued for 4 hours. The induced bacterial cells were collected, sonicated, centrifuged, and the precipitate was recovered and thoroughly dissolved using inclusion body lysis buffer. After a second centrifugation, the supernatant was collected and the recombinant protein was purified using a nickel-NTA (Ni-NTA) column. SDS-PAGE electrophoresis analysis of the purified product showed a clear protein band at approximately 55 kDa, similar to that of FliC. S.T The theoretical molecular weight of the -VP7+VP8 multi-epitope fusion protein is consistent. Figure 4 Protein concentration was determined using the BCA method, FliC. S.T The concentrations of recombinant VP7 and VP8 proteins were 1.09 mg / mL, VP7 recombinant protein was 0.68 mg / mL, and VP8 recombinant protein was 0.92 mg / mL.

[0062] Example 2 FliC S.T Immunogenicity analysis of the -VP7+VP8 multi-epitope fusion protein

[0063] (1) Mouse immunization

[0064] Twenty-five 7-week-old female BALB / c mice were randomly divided into 5 groups of 5 mice each. The mice in the first group were subcutaneously injected with 50 μg of FliC. S.T Group 1 mice received subcutaneous injections of VP7+VP8 recombinant protein; Group 2 mice received subcutaneous injections of 50 μg VP7 recombinant protein (emulsified with an equal volume of Freund's adjuvant); Group 3 mice received subcutaneous injections of 50 μg VP8 recombinant protein (emulsified with an equal volume of Freund's adjuvant); Group 4 mice received subcutaneous injections of 50 μg purified FliC. S.T Recombinant protein (Pang et al., 2024); Group 5 served as the control group, receiving a subcutaneous injection of 100 μL of sterile 0.01 M PBS buffer (pH = 7.4). After the initial immunization, booster immunizations were administered every 14 days for a total of three immunizations; Groups 2 and 3 received incomplete Freund's adjuvant during the second and third immunizations. Blood was collected via the retro-orbital vein before immunization and at days 7, 14, 21, 28, 35, and 42 post-immunization. Serum was separated and stored at -20°C. Fourteen days after the final immunization, mice were euthanized by painless cervical dislocation, and terminal serum and spleen cells were collected for subsequent cytokine level analysis.

[0065] (2) Western blot identification experiment

[0066] To further verify the function of the antigenic epitopes of the fusion protein, Western blot was used to identify its immunoreactivity. Purified VP7 or VP8 recombinant protein samples were loaded onto 12% SDS-PAGE separating gels for electrophoresis, and then transferred to PVDF membranes. The membranes were blocked overnight at 4°C using 5% skim milk powder solution. Subsequently, the PVDF membranes were separately coated with FliC diluted 1:6000. S.T Recombinant VP7+VP8 protein was used to immunize mouse serum, which was then incubated at 4°C for 1.5 h. After washing with PBST, HRP-labeled goat anti-mouse IgG (ABclonal, catalog number: AS003) diluted 1:10000 was added, and the mixture was incubated at room temperature for another 1.5 h. After multiple PBST washes, ECL was used for color development. After reacting in the dark for 90 seconds, the antigen-antibody binding signal was detected using a chemiluminescence imaging system.

[0067] The results are as follows Figure 5 As shown, both purified VP7 and VP8 recombinant proteins can be processed by FliC. S.T The specific immune serum recognition of the -VP7+VP8 recombinant protein indicates that FliC S.T The VP7+VP8 fusion protein retains the immunogenicity of both VP7 and VP8 inserted epitopes and has excellent antigen epitope display function.

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

[0069] The purified VP7 or VP8 recombinant protein was diluted with ELISA coating buffer (0.05M carbonate buffer, pH 9.6) and added to a 96-well microplate at a rate of 500 μg / well. The plate was incubated at 37°C for 1 hour, then transferred to a 4°C freezer and incubated 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 min / wash), and then 200 μL / well blocking buffer (10% skim milk powder / PBST) was added. The plate was incubated at 37°C for 1 hour. The washing process was the same as above. Mouse immune serum was serially diluted with PBST (1:200-1:12800, specifically 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800), with three replicates per dilution. 100 μL / well was incubated at 37°C for 90 min. The plate was washed three times. 100 μL / well of HRP-labeled goat anti-mouse IgG (secondary antibody, 1:5000 / PBST) was added, and the plate was incubated at 37°C in the dark for 90 min. The plate was washed three more times with PBST. 200 μL of TMB substrate was added to each well, and the plate was incubated at 37°C in the dark for 30 min. After stopping the reaction, the absorbance (OD) was measured at 650 nm. The titer of anti-VP7 and VP8 antibodies was determined by the OD of the serum sample. 650n m-value - blank control OD 650 nm Values ​​≥ 0.3 are considered positive. This value is then multiplied by the highest dilution factor of the well containing it, and finally calculated using logarithm. 10 Calculation. Result as follows: Figure 6 As shown, starting from day 7 post-immunization, FliC S.T Immunome of the -VP7+VP8 multi-epitope fusion protein can simultaneously induce high titers of anti-VP7 and anti-VP8 specific IgG, with the final antibody titer (log) 10 The values ​​were 3.28±0.13 and 3.31, respectively; while FliC S.T No corresponding antibodies were detected in the recombinant protein immunization group and the PBS control group, proving 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 day 14 post-immunization, mice were euthanized by painless cervical dislocation. The spleen was aseptically removed and placed in a sterile culture dish containing RPMI 1640 medium. The spleen was gently ground using a syringe plunger with a needle until complete tissue dissociation. The cell suspension was washed three times with RPMI 1640 and centrifuged at 1000 rpm for 10 min to collect the cell pellet. 1× erythrocyte lysis buffer (Soluble Biotech, Beijing, catalog number: R1010) was added, gently mixed, and incubated at room temperature for 5 min to allow complete lysis of the erythrocytes. The cells were then centrifuged again at 1000 rpm for 10 min, and the supernatant was discarded. The cells were resuspended in sterile PBS and centrifuged repeatedly to thoroughly remove any residual lysis buffer. The cells were then treated with RPMI 1640 containing 0.5% FBS and 1% penicillin-streptomycin antibiotics.

[0072] Cells were resuspended in 1640 medium and the cell density was adjusted to 2.5 × 10⁻⁶. 5 cells / mL. Add 2 mL of cell suspension to each well of a 6-well plate and gently agitate using the "cross-shading" method to ensure even distribution. Add purified FliC to each well to a final concentration of 5 μg / mL. S.T The VP7+VP8 recombinant protein was incubated at 37℃ in a 5% CO2 incubator for 72 hours. After incubation, 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, the levels of IFN-γ and IL-6 in the supernatant of spleen cell culture 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 beyond humoral immunity, demonstrating its good activation efficacy at the cellular immune level.

[0073] (5) Neutralizing antibody detection

[0074] In a 96-well plate, 100 μL of serum-free DMEM medium (Gibco, catalog number: 6125197) containing 10 μg / mL trypsin was added to each well. 100 μL of the five groups of mouse immune serum prepared in step (1) of Example 2 was added to the first well after sterilization, and after thorough mixing, it was continuously diluted 1:2 to a final concentration of 1:2. 11 Each dilution was replicated in four wells. Subsequently, 100 μL of a solution containing 200 TCID2 was added to each well. 50G5 and G9 PoRV virus solutions (laboratory-preserved) were incubated at 37°C for 1 hour to complete antigen-antibody pre-incubation. After incubation, MA104 cells (ATCC#CRL-2378.1) were seeded and cultured overnight in an incubator containing 5% CO2. The next day, the antibody-pre-incubated virus-serum mixture was reinfused into a new 96-well plate (100 μL / well) in the original well order. Simultaneously, normal cell control wells (8 wells, seeded with cells only) and virus control wells (100 μL per well containing 200 TCID50) were included. 50 Viral fluid, without immune serum). To assess viral titer, 100 TCID10 was also prepared in DMEM maintenance medium containing 2% FBS. 50 Virus fluid was serially diluted 10-fold (10 0 ~10 -3 100 μL per well, with 6 replicates, were used for virus regression assays. All wells were incubated at 37°C for 48 h.

[0075] After culture, the culture medium was discarded, and the plate was washed three times with PBS. 150 μL of 80% acetone pre-cooled to -20°C was added to each well, and the plate was fixed at 4°C for 30 min. The plate was then washed three times with PBS again. The plate was blocked at 37°C with 5% skim milk / PBST for 1 h, and then washed with PBST. Subsequently, 100 μL / well of the five groups of mouse immune serum prepared in step (1) of Example 2 was added to each well after 200-fold dilution and incubated at 37°C in the dark for 1 h. After washing with PBST, 100 μL / well of fluorescently labeled secondary antibody was added after 500-fold dilution and incubated at 37°C in the dark for 1 h. The plate was then washed three times with PBST. Finally, the number of infected cells in each well was observed and recorded using a fluorescence microscope, and the neutralizing antibody titer was calculated accordingly. The results are as follows: Figure 8 As shown, FliC S.T The neutralizing titers of serum from mice immunized with the -VP7+VP8 multi-epitope fusion protein against G5 and G9 PoRV virus fluids were 1:2. 9 1:2 10 This study validated its good immunogenicity, demonstrating 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 Salmonella typhimurium flagellin FliC. S.T As the backbone, the neutralizing epitopes of porcine rotavirus VP7 and VP8 proteins were replaced with the backbone protein FliC. S.T The VP7 protein was constructed using B cell epitopes with low immunogenicity and exposed on the protein surface. The amino acid sequences of the neutralizing epitopes of the VP7 protein are shown in SEQ ID NO. 1-2, and the amino acid sequences of the neutralizing epitopes of the VP8 protein are shown in SEQ ID NO. 5-6. The scaffold protein FliC... S.T The amino acid sequence is shown in SEQ ID NO. 11, 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 skeletal protein FliC is encoded. S.T The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.12, and the amino acid sequence of the multi-epitope fusion protein is shown in SEQ ID NO.

9.

2. A nucleic acid molecule encoding the multi-epitope fusion protein of claim 1, characterized in that, The DNA sequence of the nucleic acid molecule of the multi-epitope fusion protein is shown in SEQ ID NO.

10.

3. An expression box, characterized in that, Nucleic acid molecules including the multi-epitope fusion protein of claim 2.

4. A recombinant vector, characterized in that, Nucleic acid molecules including the multi-epitope fusion protein of claim 2.

5. Recombinant cells, characterized in that, Nucleic acid molecules including the multi-epitope fusion protein of claim 2.

6. A recombinant strain, characterized in that, Nucleic acid molecules including the multi-epitope fusion protein of claim 2.

7. The method for preparing the multi-epitope fusion protein according to claim 1, characterized in that, Includes the following steps: (1) Obtain nucleic acid molecules encoding neutralizing epitopes of VP7 protein and VP8 protein respectively. The nucleotide sequences of the nucleic acid molecules encoding the neutralizing epitopes of VP7 protein are shown in SEQ ID NO.3~4, and the nucleotide sequences of the nucleic acid molecules encoding the neutralizing epitopes of VP8 protein are shown in SEQ ID NO.7~8. (2) The nucleic acid molecules described in step (1) are ligated to the plasmid to obtain a recombinant expression plasmid; (3) The recombinant expression plasmid was transferred into Escherichia coli culture, induced to express, and then broken by ultrasonication, centrifuged and purified to obtain the multi-epitope fusion protein.

8. The method for preparing the multi-epitope fusion protein according to claim 4, characterized in that, The recombinant plasmid mentioned in step (1) is pUC57-FliC S.T -VP7+VP8 or pET-28a (+)-FliC S.T -VP7+VP8.

9. The use of the multi-epitope fusion protein of claim 1, the nucleic acid molecule of the multi-epitope fusion protein of claim 2, the expression cassette of claim 3, the recombinant vector of claim 4, the recombinant cell of claim 5, or the recombinant strain of claim 6 in the preparation of a drug for preventing and treating porcine rotavirus infection.

10. A vaccine or antibody, characterized in that, The vaccine comprises the multi-epitope fusion protein of claim 1; the antibody comprises the antibody induced after immunizing an animal with the multi-epitope fusion protein of claim 1.

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