Bovine parainfluenza virus 3a and 3c type multi-epitope antigen peptides, complexes and applications thereof

Abstract

The application discloses a bovine parainfluenza virus 3A and 3C type polyepitope antigen peptide, a complex thereof and application. The polyepitope antigen peptides BPMEV-3A and BPMEV-3C have amino acid sequences as shown in SEQ ID NO:1 and SEQ ID NO:3 respectively, and are connected by screening CTL epitopes, HTL epitopes and B cell epitopes from HN and F proteins of BPIV-3A and BPIV-3C strains. Animal immunization tests show that the antigen peptide and the complex thereof can effectively stimulate the body to produce specific IgG antibodies and neutralizing antibodies, induce Th1 type cellular immune response, and effectively eliminate viruses and reduce lung tissue lesions, and show good immunogenicity and protection effect. The application provides an efficient and safe vaccine candidate for prevention and control of BPIV-3, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biology and relates to multi-epitope antigenic peptides of bovine parainfluenza virus types 3A and 3C and their applications. This invention also relates to an antigenic peptide complex and its applications, especially its application in the preparation of bivalent vaccines. Background Technology

[0002] Bovine parainfluenza-3 virus (BPIV-3) belongs to the genus *Respirovirus* of the family Paramyxoviridae and is the main pathogen causing bovine parainfluenza (BPI). This disease is an acute, contagious disease that can cause respiratory illness in cattle alone, or it can cause bovine respiratory disease complex (BRDC) through mixed infections with pathogens such as *Mycoplasma bovis* and *Pasteurella multocida* type A, resulting in severe economic losses to the global cattle industry. With the large-scale development of the cattle industry, effective prevention and control of BPI is particularly important, and vaccination is currently the most effective prevention and control strategy.

[0003] BPIV-3 is an enveloped, non-segmented, single-stranded negative-sense RNA virus. Based on the nucleotide homology of the HN gene, BPIV-3 can be divided into three genotypes: A, B, and C. The predominantly circulating genotypes in my country are BPIV-3A and BPIV-3C. The HN and F proteins on the viral surface are its main antigenic proteins, which can induce the body to produce neutralizing antibodies and are the core targets for vaccine design.

[0004] Currently, commercially available BPIV-3 vaccines mainly include inactivated vaccines and live attenuated vaccines. However, these traditional vaccines have their own limitations: inactivated vaccines have good safety but poor ability to induce cellular immunity; live attenuated vaccines have strong immunogenicity but pose a potential risk of virulence reversion. In addition, because BPIV-3 is prone to gene mutation and antigenic drift under evolutionary pressure, single-subtype vaccines often cannot provide effective cross-protection against circulating strains of different subtypes.

[0005] Epitope vaccines, as a novel type of vaccine, have seen rapid development in recent years. This technology screens for multiple immunogenic epitopes in antigen proteins and tandemly constructs them into multi-epitope peptide antigens, capable of simultaneously inducing highly specific immune responses against both humoral and cellular immunity. Compared to traditional vaccines, multi-epitope vaccines offer greater design flexibility, allowing for the selection of the most protective antigenic epitopes based on the virus's evolutionary characteristics, making them particularly suitable for viruses with highly variable genomes.

[0006] Although existing studies have used bioinformatics methods to predict the antigenic epitopes of BPIV-3, most of these studies remain at the theoretical prediction stage, and no specific, experimentally validated multi-epitope antigenic peptide sequences capable of simultaneously targeting both BPIV-3A and BPIV-3C circulating strains have been published. Therefore, there is an urgent need in this field to develop a BPIV-3A and 3C bivalent multi-epitope antigenic peptide with a well-defined amino acid sequence, efficient expression in prokaryotic systems, and good immunogenicity verified in animal experiments, and to prepare a highly effective and safe vaccine based on this peptide that covers multiple subtypes.

[0007] Based on this, this invention provides two novel multi-epitope antigen peptides, BPMEV-3A and BPMEV-3C, for the first time through bioinformatics screening, molecular docking design, prokaryotic expression and purification, and animal experiments. It also confirms that the bivalent vaccine formed by the combination of the two has good immunoprotective effects and has important research value and application prospects. Summary of the Invention

[0008] The first objective of this invention is to provide a bovine parainfluenza virus type 3A and 3C multi-epitope antigen peptide.

[0009] This invention integrates CTL, HTL, and B-cell epitopes when constructing multi-epitope antigenic peptides. The synergistic effect of these three epitopes can simulate the complete pathogen infection process, comprehensively activating the body's adaptive immune system and achieving dual protection through humoral and cellular immunity. By optimizing the linker's connection method and sequence, it ensures that each epitope can be correctly processed, presented, and recognized, ultimately inducing a balanced and durable humoral and cellular immune response, providing comprehensive and effective protection for the host.

[0010] The bovine parainfluenza virus type 3A multi-epitope antigen peptide has the amino acid sequence shown in SEQ ID NO:1 and the nucleotide sequence of the encoding gene is shown in SEQ ID NO:2.

[0011] The bovine parainfluenza virus type 3C multi-epitope antigen peptide has the amino acid sequence shown in SEQ ID NO:3 and the nucleotide sequence of the encoding gene is shown in SEQ ID NO:4.

[0012] A second objective of this invention is to provide the application of the bovine parainfluenza virus type 3A and 3C multi-epitope antigenic peptides in the preparation of a kit for detecting bovine parainfluenza virus type 3.

[0013] Western blot and indirect ELISA results show that the multi-epitope antigenic peptides provided by this invention can specifically react with anti-BPIV-3 immune serum, and therefore can be used to detect bovine parainfluenza virus type 3.

[0014] A third object of the present invention is to provide a kit for detecting bovine parainfluenza virus type 3. The kit contains the aforementioned bovine parainfluenza virus type 3A or 3C multi-epitope antigenic peptide.

[0015] The fourth objective of this invention is to provide the application of the bovine parainfluenza virus type 3A and 3C multi-epitope antigenic peptides in the preparation of bovine parainfluenza virus type 3 vaccines.

[0016] Animal immunization experiments showed that both multi-epitope antigenic peptides induced specific IgG antibodies and neutralizing antibodies in guinea pigs 14 days after the initial immunization. Cytokine detection showed significantly elevated levels of IFN-γ, IL-4, and TNF-α in both immunized groups, with an IFN-γ / IL-4 ratio >1, indicating the induction of a Th1-biased mixed immune response. Challenge protection experiments showed a significant reduction in nasal viral shedding in the immunized group on day 5 after challenge, while the non-immunized group continued to shed the virus. Histopathological observation revealed only mild inflammatory cell infiltration and slight alveolar wall thickening in the lung tissue of the immunized group, while the non-immunized group showed extensive consolidation and alveolar structural destruction. In situ hybridization results showed a significantly lower viral load in the lung tissue of the immunized group compared to the non-immunized group. These results indicate that the multi-epitope antigenic peptides provided by this invention offer a safe and highly effective novel vaccine candidate for the clinical prevention and control of BPIV-3A and BPIV-3C.

[0017] A fifth objective of this invention is to provide a bovine parainfluenza virus type 3 vaccine. The vaccine comprises the bovine parainfluenza virus type 3A multi-epitope antigenic peptide or the bovine parainfluenza virus type 3C multi-epitope antigenic peptide described above.

[0018] The sixth objective of this invention is to provide an antigen peptide complex and its application.

[0019] BPMEV-3A and BPMEV-3C antigenic peptides were mixed at a 1:1 mass ratio and added to M903 oil adjuvant to prepare a bivalent vaccine with a final protein concentration of 50 μg / dose. Guinea pigs were immunized and challenged with the BPIV-3C strain via nasal drop. Specific antibody levels were detected using indirect ELISA, neutralizing antibody titers were detected using a virus neutralization assay, and the expression levels of cytokines IFN-γ, IL-4, TNF-α, and IL-10 were detected using ELISA. Viral shedding from nasal swabs after challenge was monitored by RT-qPCR, and lung tissue pathological changes and viral load were evaluated by HE staining and in situ hybridization. The results showed that the bivalent vaccine not only induced the production of specific IgG antibodies and neutralizing antibodies, but also that the neutralizing antibody titer after the second immunization was significantly higher than that of the monovalent vaccine group, indicating that it induced a more durable and efficient immune response. The bivalent vaccine was comparable to the monovalent vaccine in terms of virus clearance rate and lung tissue protection, but it could simultaneously target both bovine parainfluenza virus subtypes 3A and 3C, providing a broader spectrum of protection.

[0020] The beneficial effects of this invention are:

[0021] This invention provides the first-ever multi-epitope antigenic peptides and their encoding genes targeting prevalent BPIV-3A and BPIV-3C strains. The proteins, purified using a prokaryotic expression system, specifically bind to anti-BPIV-3 immune serum, exhibiting good reactivity. These antigenic peptides induce high levels of specific IgG antibodies and neutralizing antibodies in guinea pigs, triggering a Th1-biased mixed immune response, achieving dual activation of humoral and cellular immunity. This results in excellent protection against viral challenge, with broad-spectrum protection and high safety. This invention provides a highly effective and safe vaccine candidate for the prevention and control of BPIV-3, demonstrating promising application prospects. Attached Figure Description

[0022] Figure 1 : Dating models of vaccine proteins with TLR4 receptor molecules. A: Dating model of BPMEV-3A with TLR4 receptor molecules; B: Dating model of BPMEV-3C with TLR4 receptor molecules.

[0023] Figure 2 PAGE results of vaccine protein expression and purification. A: PAGE results of BPMEV-3A protein expression and purification; B: PAGE results of BPMEV-3C protein expression and purification; M is the molecular weight standard of protein; Lane 1 is the bacterial lysate precipitate; Lane 2 is the loading solution; Lane 3 is the flow-through solution; Lane 4 is the protein washing solution; Lanes 5-11 are the target protein elution solution.

[0024] Figure 3 : In vitro immunoreactivity verification. A: Western blot results of BPMEV-3A, BPMEV-3C and guinea pig anti-BPIV-3 immune serum (primary antibody); B: Western blot results of BPMEV-3A, BPMEV-3C and guinea pig negative serum (primary antibody).

[0025] Figure 4 : Specific ELISA antibody titer of guinea pig immune serum.

[0026] Figure 5 Neutralizing antibody titers in guinea pig immune serum.

[0027] Figure 6 ELISA was used to detect the protein expression levels of cytokines IFN-γ, IL-4, TNF-α, and IL-10 in guinea pig immune serum.

[0028] Figure 7: Pathological sections of guinea pig lung tissue after challenge. In the figure, Control group is the control group; BPIV-3A challenge is the BPIV-3A non-immune challenge group; BPIV-3C challenge is the BPIV-3C non-immune challenge group; BPMEV-3A group is the BPMEV-3A monovalent vaccine challenge group; BPMEV-3C group is the BPMEV-3C monovalent vaccine challenge group; BPMEV-AC group is the BPMEV-3A and BPMEV-3C bivalent vaccine challenge group.

[0029] Figure 8 : In situ hybridization results of guinea pig lung sections after challenge. In the figure, Control group is the control group; BPIV-3A challenge is the BPIV-3A non-immunized challenge group; BPIV-3C challenge is the BPIV-3C non-immunized challenge group; BPMEV-3A group is the BPMEV-3A monovalent vaccine immunized challenge group; BPMEV-3C group is the BPMEV-3C monovalent vaccine immunized challenge group; BPMEV-AC group is the BPMEV-3A and BPMEV-3C bivalent vaccine immunized challenge group. DAPI: Nuclear staining (blue fluorescence), showing tissue cell distribution; CY3: BPIV-3 viral RNA probe labeling (red fluorescence), showing viral distribution and viral load; Merge: Fusion diagram, showing the relative positional relationship between the virus and cells. Detailed Implementation

[0030] The present invention will now be described in detail through specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and not for limiting the scope of protection of the present invention. Various modifications or equivalent substitutions made by those skilled in the art based on the following embodiments should also be considered to fall within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods and conditions, or selected according to the product instructions. Unless otherwise specified, the experimental materials involved in the present invention are conventional reagents and conditions in the art.

[0031] Key materials description: pET-MBP plasmid: a prokaryotic soluble secretory expression vector, a commercially available vector, preserved in our laboratory; BPIV-3A and BPIV-3C strains: isolated and preserved in our laboratory.

[0032] Example 1: Design of a multi-epitope vaccine

[0033] 1. Candidate antigen proteins for vaccine design

[0034] Using the BPIV-3 antigen protein as a target, the HN and F proteins of BPIV-3A and BPIV-3C were obtained from the National Center for Biotechnology Information (NCBI) database. The downloaded protein sequences were analyzed using Geneious Prime software, and conserved peptides were screened.

[0035] 2. Prediction of transmembrane domains of antigen proteins

[0036] The amino acid sequences of the HN and F proteins of BPIV-3A were obtained from GenBank: AEU04140.1 and GenBank: AEU04139.1, respectively; the amino acid sequences of the HN and F proteins of BPIV-3C were obtained from GenBank: WPR21741.1 and GenBank: WPR21740.1, respectively. The extracellular domains of these antigen proteins were predicted using the TMHMM-2.0 online database. The results showed that the extracellular domain of the HN protein in both BPIV-3A and BPIV-3C was 57aa-572aa; and the extracellular domain of the F protein in both BPIV-3A and BPIV-3C was 1aa-493aa.

[0037] 3. T cell and B cell epitope prediction

[0038] The online website NetBoLApan-1.0 was used to predict CTL epitopes of the HN and F proteins of BPIV-3A and BPIV-3C, with an amino acid length set to 9 aa; the online website NetBoLAIIpan-1.0 was used to predict HTL epitopes of the HN and F proteins of BPIV-3A and BPIV-3C, with an amino acid length set to 15 aa. In predicting T cell epitopes, this experiment aimed to increase the number of BoLA class I and BoLA class II alleles selected and to use high-frequency epitopes as candidates, thereby expanding the vaccine's coverage across different cattle breeds and thus broadening its protective range.

[0039] Linear B-cell epitopes of the extracellular domains of selected antigen proteins were predicted using the ABCpred online website, SVMTriP, and IEDB online website, with overlapping epitopes used as candidate epitopes.

[0040] Epitopes for CTL, HTL, and linear B cells were predicted for antigenicity using the VaxiJen online website; epitope toxicity was predicted using the ToxinPred online website; and epitope allergenicity was predicted using the AllerTOP v2.1 online website. Epitopes with high conservation, good antigenicity, no toxicity, and no allergenicity were ultimately selected. The final CTL, HTL, and linear B cell epitopes selected are shown in Table 1.

[0041] Table 1. CTLEs, HTLEs, and BE sequences

[0042]

[0043] 4. Construction of multi-epitope vaccines

[0044] Connect the filtered T-tablets and B-tablets using different linkers.

[0045] (1) The CTL epitopes of the F and HN proteins of BPIV-3A in Table 1 were linked together using linker-AAY. The amino acid sequence of the CTL epitope of BPIV-3A after linking is as follows:

[0046] YSITLQVRL AAY KSMKISQNF AAY KGIDTTFS AAY RSTSWHSKL

[0047] (2) The HTL epitopes of the F and HN proteins of BPIV-3A in Table 1 were linked together using linker-GPGPG. The amino acid sequence of the HTL epitope of BPIV-3A after linking is as follows:

[0048] YDGLKLQKDVIVVNH GPGPG ETRYLILSLIPKIEN GPGPG LQIGIITINSDLVPD GPGPG NPSLTSSPKIRLIPG

[0049] (3) The linear B-cell epitopes of the F and HN proteins of BPIV-3A in Table 1 were linked together using linker-KK. The amino acid sequence of the linear B-cell epitope of BPIV-3A after linking is as follows:

[0050] TESIKMRVIDVDLS KK ITEIFTTSTVDQYDIYDL KK FSSYICPSDPGFILN KK THNNTGLRTKR KK VLQIGIITINSDLV KK NRKSCSLALLNT KK TQGCQDIGKSYQVLQI KK CPGKTQRDC KK ELAIYNRTLPAAYTTT KK THDRGIEPLNPDNF

[0051] (4) The CTL epitopes of the F and HN proteins of BPIV-3C in Table 1 were linked together using linker-AAY. The amino acid sequence of the CTL epitope of BPIV-3C after linking is as follows:

[0052] AQITAAVAL AAY AGLQLGIAL AAY KGMKISQNF AAY VQNYIPLSL AAY KGVDTTFNL

[0053] (5) The HTL epitopes of the F and HN proteins of BPIV-3C in Table 1 were linked together using linker-GPGPG. The amino acid sequence of the HTL epitope of BPIV-3C after linking is as follows:

[0054] YDGLKLQKDVIVVRH GPGPG ENPIITYATDTRRVN GPGPG GYCFHIVEINHRSLD

[0055] (6) The linear B-cell epitopes of the F and HN proteins of BPIV-3C in Table 1 were linked together using linker-KK. The amino acid sequence of the linear B-cell epitope of BPIV-3C after linking is as follows:

[0056] DLLFTESIKMRVIDVDLN KK ESNNSTSSRKKRF KK PNKEGTLATY KK PNHIMTKGAFLGGADI KK KVDERSDYASTGI KK LANKRDQQ KK PIQRMTHDSGIEPLNP KK CPGKTQRDC KK NHNKIASQQMRREFAE

[0057] (7) Use linker-EAAAK to link bovine β-defensin-3 sequence to CTL epitope, use linker-GPGPG to link CTL epitope to HTL epitope; use linker-KK to link HTL epitope to B cell epitope; use linker-GSGS to link linear B cell epitope to strepII sequence (WSHPQFEK, tag for subsequent protein expression and purification).

[0058] Among them, bovine β-defensin-3 sequence, as an immunostimulatory factor, can enhance immunogenicity and improve immune effect when inserted into epitope sequence. Its amino acid sequence is: MRLHHLLLALLFLVLSAGSGFTQGARNHVTCRINRGFCVPIRCPGRTRQIGTCFGPRIKCCRSW.

[0059] The experiment ultimately designed two vaccine constructs, named “BPMEV-3A” (amino acid sequence as shown in SEQ ID NO:1) and “BPMEV-3C” (amino acid sequence as shown in SEQ ID NO:3), with the nucleotide sequences of the encoding genes shown in SEQ ID NO:2 and SEQ ID NO:4, respectively.

[0060] BPMEV-3A amino acid sequence (SEQ ID NO:1): MRLHHLLLALLFLVLSAGSGFTQGARNHVTCRINRGFCVPIRCPGRTRQIGTCFGPRIKCCRSW EAAAK YSITLQVRLAAYKSMKISQNFAAYKGIDTTFSAAYRSTSWHSKL GPGPG YDGLKLQKDVIVVNHGPGPGETRYLILSLIPKIENGPGPGLQIGIITINSDLVPDGPGPGNPSLTSSPKIRLIPG KK TESIKMRVIDVDLSKKITEIFTTSTVDQYDIYDLKKFSSYICPSDPGFILNKKTHNNTGLRTKRKKVLQIGIITINSDLVKKNRKSCSLALLNTKKTQGCQDIGKSYQVLQIKKCPGKTQRDCKKELAIYNRTLPAAYTTTKKTHDRGIEPLNPDNF GSGS WSHPQFEK

[0061]

[0062] BPMEV-3C amino acid sequence (SEQ ID NO:3): MRLHHLLLALLFLVLSAGSGFTQGARNHVTCRINRGFCVPIRCPGRTRQIGTCFGPRIKCCRSW EAAAK AQITAAVALAAYAGLQLGIALAAYKGMKISQNFAAYVQNYIPLSLAAYKGVDTTFNL GPGPG YDGLKLQKDVIVVRHGPGPGENPIITYATDTRRVNGPGPGGYCFHIVEINHRSLD KK DLLFTESIKMRVIDVDLNKKESNNSTSSRKKRFKKPNKEGTLATYKKPNHIMTKGAFLGGADIKKKVDERSDYASTGIKKLANKRDQQKKPIQRMTHDSGIEPLNPKKCPGKTQRDCKKNHNKIASQQMRREFAE GSGS WSHPQFEK

[0063]

[0064] 5. Prediction of the physicochemical properties of multiepitope vaccines

[0065] The physicochemical properties of BPMEV-3A and BPMEV-3C were predicted using the Expasy ProtParam online website. The results showed that BPMEV-3A protein had an antigenicity of 0.6404, an instability index of 25.24, a lipid index of 85.19, a hydrophilicity of -0.368, and a solubility of 0.579; while BPMEV-3C protein had an antigenicity of 0.5680, an instability index of 29.34, a lipid index of 74.03, a hydrophilicity of -0.538, and a solubility of 0.518. This indicates that both BPMEV-3A and BPMEV-3C proteins possess good antigenicity, stability, hydrophilicity, and solubility.

[0066] 6. Molecular docking

[0067] The bovine TLR4 receptor plays a crucial role in pathogen recognition and innate immune activation, and is a key target in vaccine immunization. This experiment used the HDOCK online database to predict the docking of BPMEV-3A and BPMEV-3C proteins with the bovine TLR4 receptor. Figure 1 A is the docking complex of BPMEV-3A and TLR4 receptor, with a docking energy of -306.74 kcal / mol; Figure 1 B is the docking complex of BPMEV-3C and the TLR4 receptor, with a docking energy of -334.24 kcal / mol, indicating that BPMEV-3A and BPMEV-3C proteins have a strong binding affinity to the bovine TLR4 receptor.

[0068] Example 2: Preparation of a multi-epitope vaccine

[0069] 1. Construction of recombinant plasmids pET-MBP-BPMEV3A and pET-MBP-BPMEV3C

[0070] First, the pET-MBP empty vector plasmid was double-digested with XhoⅠ (NEB, #R0146V) and HindⅢ (NEB, #R0104V). The digestion reaction volume was: XhoⅠ: 1 μL, HindⅢ: 1 μL, NEBuffer™ r2.1: 5 μL, plasmid DNA: 1 μg, and ddH2O to a final volume of 50 μL. The digestion conditions were 37℃ for 3 h. The digestion products were then purified by gel extraction.

[0071] The amino acid sequences of BPMEV-3A and BPMEV-3C were sent to the company for codon optimization and gene synthesis. Using the synthesized plasmid pET28a-sumo-BPMEV3A as an amplification template, the target fragment BPMEV-3A was amplified using upstream and downstream primers containing the pET-MBP empty homologous arm; similarly, using the synthesized plasmid pET28a-sumo-BPMEV3C as an amplification template, the target fragment BPMEV-3C was amplified using upstream and downstream primers containing the pET-MBP empty homologous arm. The PCR amplification products were purified by gel extraction.

[0072] The linearized vector and the target fragment were ligated using homologous recombination. The reaction mixture consisted of 5 μL of 2×CE Mix (Novizan, #C116), 4 μL of linearized vector, and 1 μL of insert fragment. The reaction conditions were 50 °C for 5 min. Recombinant plasmids pET-MBP-BPMEV3A and pET-MBP-BPMEV3C were constructed and transformed into *E. coli* DH5α competent cells. Several positive clones were selected and transferred to 5 ml of liquid LB medium containing ampicillin for amplification. 300 μL of the bacterial culture was sent to Kexin Technology Co., Ltd. for sequencing. The correctly sequenced bacterial cultures were stored at -80 °C, and plasmids were extracted after amplification of the corresponding bacterial cultures.

[0073] 2. Expression and identification of recombinant BPMEV-3A and BPMEV-3C proteins

[0074] Recombinant plasmids pET-MBP-BPMEV3A and pET-MBP-BPMEV3C were transformed into Escherichia coli BL21(DE3) competent cells. Positive clones were selected and transferred to 8 ml of liquid LB medium containing ampicillin for expansion culture. The culture was carried out at 37°C and 180 rpm for 12-16 h on a shaker and then stored at -80°C as seed culture.

[0075] Two recombinant expression bacteria were inoculated at a ratio of 1:100 into 1L of liquid LB medium containing ampicillin and cultured at 37°C and 180rpm until OD reached... 600nm When the bacterial cell viscosity reached 0.4-0.6, IPTG was added to a final concentration of 0.5 mM, and expression was induced at 16℃ and 160 rpm for 18 h. The induced bacterial cell pellet was collected, high-pressure disrupted, and centrifuged to obtain the supernatant. The protein was purified using a Strep II affinity chromatography column. The purified sample was validated by SDS-PAGE, and the results are as follows: Figure 2 As shown in Figures A and 2B, since the MBP tag element size is approximately 44 kDa, the final band size of BPMEV-3A is approximately 84 kDa, and the band size of BPMEV-3C is approximately 81 kDa. Both recombinant proteins were then concentrated to 1 ml by ultrafiltration. After determining the protein concentration, they were stored at -80°C for later use.

[0076] Further Western blotting analysis was performed to assess the reactivity of BPMEV-3A and BPMEV-3C proteins. Purified BPMEV-3A and BPMEV-3C protein samples were incubated with anti-BPIV-3 immune serum (guinea pig source) and guinea pig negative serum, respectively, to evaluate the specific binding ability of these two epitope proteins to anti-BPIV-3 immune serum. Results are as follows: Figure 3 As shown, Figure 3 A represents the results of incubation of the two proteins with anti-BPIV-3 immune serum. Figure 3 B shows the results of incubation of the two proteins with guinea pig negative serum. The results indicate that both BPMEV-3A and BPMEV-3C proteins can be specifically recognized by BPIV-3 immune serum, suggesting that the two epitope proteins are reactive.

[0077] 3. Identification of the in vitro immunoreactivity of BPMEV-3A and BPMEV-3C recombinant proteins

[0078] The in vitro immunoreactivity of BPMEV-3A and BPMEV-3C recombinant proteins was analyzed using an indirect ELISA method. Purified BPMEV-3A and BPMEV-3C recombinant proteins were used as coating antigens and coated onto 96-well ELISA plates at a concentration of 2 μg / ml, incubated overnight at 4°C. Anti-BPIV-3 immune serum (guinea pig source) was used as the primary antibody for incubation, and guinea pig negative serum (primary antibody) served as a negative control. Serum was serially diluted (1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, 1:204800), and goat anti-guinea pig IgG-HRP was used as the secondary antibody. The results showed that BPMEV-3A (Table 2) and BPMEV-3C recombinant proteins (Table 3) specifically bound to anti-BPIV-3 guinea pig hyperimmune serum, with titers reaching 1:25600, demonstrating good in vitro immunoreactivity.

[0079] Table 2. Indirect ELISA detection of BPMEV-3A protein immunoreactivity

[0080]

[0081] Table 3. Immunoreactivity of BPMEV-3C protein detected by indirect ELISA

[0082]

[0083] Example 3: Vaccine preparation and guinea pig experiments

[0084] 1. Preparation of multi-epitope vaccines

[0085] BPMEV-3A and BPMEV-3C vaccine proteins (adjuvant:vaccine protein mass ratio of 1:1) were added to M903 oil adjuvant (produced by Chengdu Yisikang Biotechnology Co., Ltd.) to make the final concentration of vaccine protein 50 μg / dose, of which 25 μg / dose of BPMEV-3A and BPMEV-3C were added to the bivalent vaccine. After emulsification and quality inspection, the vaccine was stored at 4℃ in the dark.

[0086] 2. Guinea pig animal experimental protocol

[0087] Twenty-four SPF-grade female guinea pigs weighing 300-350g were randomly divided into six groups of four each: three immune challenge groups (BPMEV-3A protein vaccine group, BPMEV-3C protein vaccine group, and combined immunization group of the two proteins), two non-immunized challenge groups, and one blank control group. Guinea pigs were immunized via intramuscular injection in the leg, while the blank control group received the same volume of PBS. The immunized groups received three immunizations, with booster immunizations every 14 days after the initial immunization. Fourteen days after the third immunization, the guinea pigs were challenged via nasal drops. Blood was collected from the anterior vena cava before immunization (day 0), and at 14, 28, and 42 days after the initial immunization. The guinea pigs were sacrificed 14 days after challenge, and the serum was collected and stored at -20°C.

[0088] Table 4 Animal Experimentation Protocol

[0089]

[0090] 3. Specific IgG antibody detection

[0091] The level of IgG antibodies in the serum of immunized guinea pigs was detected by indirect ELISA. BPIV-3A and BPIV-3C viral proteins preserved in this laboratory were used as coating antigens, coated onto 96-well ELISA plates at a concentration of 2 μg / ml, and incubated overnight at 4°C. Serum from guinea pigs before immunization and at 14, 28, and 42 days after the first immunization was used as the primary antibody for incubation. Guinea pig negative serum (primary antibody) was used as a negative control. Serum was serially diluted (1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, 1:204800), and goat anti-guinea pig IgG-HRP was used as the secondary antibody for incubation.

[0092] The results are as follows Figure 4 As shown, the levels of specific antibodies in the BPMEV-3A, BPMEV-3C, and BPMEV-AC immunization groups significantly increased after the first immunization. These results indicate that the developed vaccine can effectively induce the production of specific antibodies in animals after immunization, demonstrating its good immunogenicity.

[0093] 4. Neutralizing antibody detection

[0094] After inactivating the serum sample at 56℃ for 30 minutes, it was serially diluted (1:2). 1 To 1:2 8 Each dilution was replicated in 3 wells. 50 μL of the virus was added to each well of a 96-well plate, and the BPIV-3 virus was diluted to 100 TCID using DMEM. 50 Add 50 μL of serum to each well containing different dilutions. A normal cell control group and a virus control group were also included. After incubating the inoculated plates at 37°C for 1 hour, supplement each well with serum at a concentration of 2–5 × 10⁻⁵. 5 Add 100 μL of MDBK cell suspension per cell / mL, mix well, and continue culturing for 3–5 days, observing cytopathic effects. Results are considered valid only when the virus control shows typical cytopathic effect (CPE), positive serum completely inhibits viral CPE, negative serum shows no inhibitory effect, and the cell control is normal. Neutralizing antibody titers are calculated using the Reed-Muench method, with the highest serum dilution capable of inhibiting 50% cytopathic effects representing the antibody titer.

[0095] The results are as follows Figure 5 As shown, no neutralizing antibodies were detected in the blank control group before and after immunization, while the neutralizing titer in the BPMEV-3A monotherapy group was 10 on day 14 after the first immunization. -0.9 The neutralizing titer reached its maximum of 10 on day 14 (42 days) after the third vaccination. -1.071 In the BPMEV-3C monotherapy group, the neutralizing titer rose to 10 mmol / L 14 days after the first immunization. -1.628 Antibody titers 14 days after the second and third immunizations were almost the same as those 14 days after the first immunization. In the BPMEV-AC bivalent vaccine immunization group, the neutralizing antibody titer reached 10 mmol / L 14 days after the first immunization. -1.567 The neutralizing antibody titer reached its peak of 10 on day 14 (28 days) after the second immunization. -1.945 The neutralizing antibody titers in the BPMEV-AC bivalent vaccine immunization group after the second immunization were generally higher than those in the BPMEV-3A and BPMEV-3C monovalent vaccine immunization groups. These results indicate that BPMEV-3A and BPMEV-3C multi-epitope vaccines, as well as the BPMEV-AC bivalent multi-epitope vaccine, provide good immunoprotective effects, with the BPMEV-AC bivalent multi-epitope vaccine offering superior protection.

[0096] 5. Cytokine detection

[0097] The expression levels of IFN-γ, IL-4, TNF-α, and IL-10 in the serum of guinea pigs were detected by ELISA at 14, 28, and 42 days after the first immunization. The specific steps were performed in accordance with the instructions of the ELISA kit.

[0098] The results are as follows Figure 6As shown, the levels of IFN-γ secreted by Th1 cells in all groups significantly increased after immunization, suggesting that the multi-epitope vaccine can stimulate the body to produce a cellular immune response and effectively resist intracellular pathogens. The levels of IL-4 secreted by Th2 cells showed an increasing trend after immunization, with an IFN-γ / IL-4 ratio >1; overall, the response still leaned towards a Th1-type mixed response. The pro-inflammatory cytokine TNF-α and the anti-inflammatory cytokine IL-10 also showed an increasing trend, but the differences were not significant, suggesting that the multi-epitope vaccine has a controlled immune response, and the system initiates protective regulatory mechanisms while launching an attack. The results indicate that the BPMEV-3A and BPMEV-3C multi-epitope vaccines can effectively stimulate guinea pigs to produce high levels of cytokines and induce a good cellular immune response.

[0099] 6. In vitro detoxification monitoring

[0100] To evaluate the protective efficacy of BPMEV-3A and BPMEV-3C vaccine proteins, BPIV-3A and BPIV-3C strains were administered via nasal drops. Nasal swabs were collected daily from day 1 to day 7 post-challenge, and in vitro viral shedding was detected using RT-qPCR. The results are shown in Table 5. Nasal viral shedding decreased in the vaccine group starting on day 5 post-challenge, while no obvious signs of viral clearance were observed in the non-immunized group.

[0101] Table 5. In vitro detoxification monitoring

[0102]

[0103] 7. Pathological tissue observation

[0104] Fourteen days after the viral challenge, an autopsy was performed to observe the lung lesions, and lung tissue sections were prepared for hematologic staining (HE). The results were as follows: Figure 7 As shown, the lung tissue structure in the blank group was normal, with no obvious inflammatory cell infiltration; lung sections in the non-immunized group showed extensive consolidation, alveolar stenosis or even disappearance, granulocyte exudation in the alveolar cavity, and exfoliated epithelial cells in the cavity; alveolar walls in the immune challenge group showed small-scale mild thickening, with occasional inflammatory cell infiltration.

[0105] 8. Detection of viral load in lung tissue

[0106] To evaluate the protective effect of the vaccine, antisense RNA probes BPIV-3A and BPIV-3C were designed and hybridized to slides to visualize the expression abundance of RNA in lung tissue in situ. The results are as follows: Figure 8 As shown, the fluorescence signal intensity in the lungs of the non-immune challenge group was higher and more uniform, indicating a more widespread viral infection; the fluorescence signal of the immune challenge group and the non-immune challenge group showed a significant difference, indicating that the immune response in this area had a virus clearance effect.

[0107] In summary, the multi-epitope vaccines designed in this embodiment have demonstrated good immunogenicity through in vitro and in vivo experiments, confirming that the BPMEV-3A and BPMEV-3C multi-epitope vaccines have good application potential as novel vaccines.

Claims

1. A bovine parainfluenza virus type 3A multi-epitope antigenic peptide, having an amino acid sequence as shown in SEQ ID NO:

1. 2.A gene encoding the bovine parainfluenza virus type 3A multi-epitope antigenic peptide of claim 1, having a nucleotide sequence as shown in SEQ ID NO:

2. 3.A bovine parainfluenza virus type 3C multi-epitope antigenic peptide, having an amino acid sequence as shown in SEQ ID NO:

3. 4.A gene encoding the bovine parainfluenza virus type 3C multi-epitope antigenic peptide of claim 3, having a nucleotide sequence as shown in SEQ ID NO:

4. 5.An antigenic peptide complex comprising the bovine parainfluenza virus type 3A multi-epitope antigenic peptide of claim 1 and the bovine parainfluenza virus type 3C multi-epitope antigenic peptide of claim 3. 6.Use of the antigenic peptide of claim 1 or 3 or the antigenic peptide complex of claim 5 in the preparation of a kit for detecting bovine parainfluenza virus type 3. 7.A kit for detecting bovine parainfluenza virus type 3, comprising the antigenic peptide of claim 1 or 3 or the antigenic peptide complex of claim 5. 8.Use of the antigenic peptide of claim 1 or 3 or the antigenic peptide complex of claim 5 in the preparation of a bovine parainfluenza virus type 3 vaccine. 9.A bovine parainfluenza virus type 3 vaccine, comprising the antigenic peptide of claim 1 or 3 or the antigenic peptide complex of claim 5.

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