Subunit vaccine for porcine reproductive and respiratory syndrome virus

Abstract

The invention relates to the technical field of biology, in particular to a porcine reproductive and respiratory syndrome virus subunit vaccine. Through bioinformatics analysis and experimental verification, the antigen protein with excellent immunogenicity is obtained through screening, and the amino acid sequence of the antigen protein is shown as SEQ ID NO: 1. The subunit vaccine constructed on the basis of the protein can effectively induce mice to generate high-level neutralizing antibodies, the effect of the subunit vaccine is remarkably better than that of commercial control vaccines, the mice can be stimulated to generate strong humoral immunity and cellular immunity at the same time, and the induced immune response type is mainly Th2 type. Further research shows that the antigen protein and the virus structural protein E + M are combined for immunization, and the immune effect of the structural protein can be remarkably enhanced. The subunit vaccine developed by the invention provides a new thought for research and development of novel vaccines for resisting PRRSV and other viral diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a subunit vaccine against porcine reproductive and respiratory syndrome virus (PRRSV). Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a serious infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). Since its first report in my country, the circulating PRRSV strains have undergone significant evolution, gradually developing from classic strains to highly pathogenic strains, and further evolving into strains such as NADC30. This virus can cause reproductive disorders in pregnant sows and respiratory diseases in piglets, resulting in continuous economic losses to the pig farming industry.

[0003] Currently, the prevention and control of PRRS mainly relies on two types of vaccines: live attenuated vaccines (MLV) and inactivated vaccines. However, existing vaccine technologies still have multiple limitations and are difficult to meet actual prevention and control needs. Although live attenuated vaccines can induce a certain degree of humoral and cellular immune responses, their cross-protective effect against heterologous strains is limited. When such vaccines persist in immunized pig herds for a long time, they may pose safety risks through virulence reversion, viral recombination, or gene mutation. Furthermore, the persistence of the virus may promote vertical or horizontal transmission, exacerbating the genetic diversity of strains in the field. Although inactivated vaccines have outstanding safety profiles, they often fail to effectively stimulate cellular immune responses and neutralizing antibody responses, resulting in poor overall protective efficacy. Studies have shown that while inactivated vaccines can partially alleviate clinical symptoms in PRRSV-positive fields, they cannot block virus transmission and may cause adverse reactions such as fever and decreased feed intake in immunized pig herds.

[0004] The PRRSV genome is highly mutable, and its complex envelope protein structure leads to enhanced concealment of neutralizing epitopes. Simultaneously, the virus can evade the immune system by inducing immunosuppressive factors such as IL-10. Existing vaccine platforms face technical bottlenecks in coordinating humoral and cellular immunity, making it difficult to simultaneously meet the comprehensive requirements of safety, clear composition, and strong immune response. Therefore, there is an urgent need to develop a novel porcine reproductive and respiratory syndrome virus subunit vaccine with high safety, clear composition, and the ability to induce a strong immune response and provide protective efficacy, providing more effective technical support for PRRS prevention and control. Summary of the Invention

[0005] In view of this, the present invention proposes a subunit vaccine for porcine reproductive and respiratory syndrome virus.

[0006] The technical solution of this invention is implemented as follows:

[0007] In a first aspect, the present invention provides a porcine reproductive and respiratory syndrome virus antigen, wherein the antigen (i.e., recombinant protein NSP2) 236The amino acid sequence of the antigen is shown in SEQ ID NO:1. Further, the nucleotide sequence of the gene encoding the antigen is shown in SEQ ID NO:2.

[0008] Secondly, the present invention provides the application of the antigen in the preparation of products for the prevention and treatment of porcine reproductive and respiratory syndrome.

[0009] Thirdly, the present invention provides an antigen combination comprising an antigen protein with an amino acid sequence as shown in SEQ ID NO:1.

[0010] Furthermore, in some specific embodiments, the antigen combination further includes a fusion protein formed by linking peptides of the E protein and M protein of porcine reproductive and respiratory syndrome virus.

[0011] Fourthly, the present invention provides the application of the antigen combination described above in the preparation of products for the prevention and treatment of porcine reproductive and respiratory syndrome.

[0012] Based on the antigen combination, the present invention also provides a porcine reproductive and respiratory syndrome virus vaccine, the vaccine comprising a first antigen, a second antigen and a vaccine adjuvant; the amino acid sequence of the first antigen is shown in SEQ ID NO:1, the second antigen is an antigen protein (i.e., E+M protein) with an amino acid sequence shown in SEQ ID NO:11; the volume ratio of the first antigen, the second antigen and the vaccine adjuvant is 1:1:1.

[0013] Fifthly, the present invention provides a porcine reproductive and respiratory syndrome virus subunit vaccine, the vaccine comprising an antigen protein with an amino acid sequence as shown in SEQ ID NO:1 and a vaccine adjuvant.

[0014] Furthermore, the adjuvant is Freund's adjuvant.

[0015] Furthermore, the volume ratio of the adjuvant to the antigen is 1:1.

[0016] In a sixth aspect, the present invention provides an expression vector comprising a gene fragment with a nucleotide sequence as shown in SEQ ID NO:2.

[0017] The beneficial effects of the present invention include at least the following:

[0018] (1) This invention selects the non-structural protein NSP2 as the target, instead of the conventional use of viral structural proteins as the target, and through bioinformatics analysis and experimental verification, accurately locates a B cell epitope fragment NSP2 that can effectively stimulate protective humoral immunity from NSP2. 236 The experimental results show that NSP2 236As an antigen, it can induce the body to produce high-titer neutralizing antibodies. Subunit vaccines, due to their high safety profile, often have relatively weak immunogenicity and are difficult to induce very high levels of neutralizing antibodies. However, the NSP2 of this invention... 236 The subunit vaccine produced a neutralizing antibody titer of 1:107 in mice, significantly higher than the commercial vaccine control group (1:39). The subunit vaccine technology employed in this invention contains only recombinant B-cell epitope fragments and no infectious components such as viral nucleic acids, resulting in high safety and overcoming the common drawback of weak immunogenicity in traditional subunit vaccines. Animal experiments demonstrate that this vaccine not only elicits strong humoral immunity but also induces a significant cellular immune response, achieving an ideal effect with both excellent humoral and cellular immune responses.

[0019] (2) This invention will recombinant NSP2 236 Combined immunization with viral structural protein E+M significantly enhanced the immune effect of the structural protein. The results showed that the combined immunization group induced higher levels of cellular and humoral immunity and produced more durable and higher levels of neutralizing antibodies.

[0020] (3) This invention constructs NSP2 236 Prokaryotic expression vector, expressing NSP2 via E. coli expression system 236 Protein, NSP2 236 The preparation of subunit vaccines after purification has advantages such as low cost, short cycle, and easy scale-up production, providing important theoretical basis and technical support for the research and development of PRRSV and other viral subunit vaccines. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The recombinant protein NSP2 of this invention 236 The results of prokaryotic expression experiments are shown, where: A is the double enzyme digestion identification of the recombinant plasmid; B is NSP2. 236 The expression of the target protein is indicated by the red box; C represents NSP2. 236 Electrophoresis results of the purified product; D represents NSP2. 236 Western blotting reaction bands with His-tagged antibodies; E represents NSP2. 236 Western blotting reaction bands with PRRS-positive serum;

[0023] Figure 2 The recombinant protein NSP2 of this invention 236 Safety evaluation process and related experimental results after immunization of mice, including: A is the vaccination protocol and sampling flowchart; B is the change in mouse body weight within 7 days after the first immunization; C is the detection of the change in mouse body weight within 7 days after the second immunization; D is the pathological changes in spleen tissue sections.

[0024] Figure 3 The recombinant protein NSP2 of this invention 236 The results of cytokine expression levels in spleen lymphocytes after immunization of mice, where: A represents the expression level of IL-4 mRNA; B represents the expression level of IL-6 mRNA; C represents the expression level of IL-10 mRNA; and D represents the expression level of TNF-α mRNA.

[0025] Figure 4 The recombinant protein NSP2 of this invention 236 Results of cellular immunity levels in immunized mice, where: A is the result of ELISpot assay; B is the expression level of IFN-γ in splenic lymphocytes; C is the result of lymphocyte proliferation activity assay 42 days after immunization.

[0026] Figure 5 The recombinant protein NSP2 of this invention 236 Results of humoral immunity levels in mice after immunization, where: A represents the level of specific antibody IgG; B represents the level of specific antibody IgG1; C represents the level of specific antibody IgG2a; D represents NSP2. 236 Analysis of the results of inducing immune bias;

[0027] Figure 6 The recombinant protein NSP2 of this invention 236 Results of neutralizing antibody levels in mice after immunization;

[0028] Figure 7 The recombinant protein NSP2 of this invention 236 Immunohistochemical results after immunizing mice, where: A is the immunohistochemical result of mouse spleen (20×); B is CD3... + Percentage of T-cell positive area; C represents CD4. + The percentage of T-cell positive area; D represents CD8. + Percentage of T-cell positive area; E CD4 + T cells and CD8 + T cells relative to CD3 + Changes in the ratio of T cells;

[0029] Figure 8 NSP2 is the present invention236 Safety evaluation process and related experimental results after combined immunization of mice with structural protein E+M, including: A is the vaccination protocol and sampling flowchart; B is the change in mouse body weight within 7 days after the first immunization; C is the detection of the change in mouse body weight within 7 days after the second immunization; D is the pathological changes in spleen tissue sections.

[0030] Figure 9 NSP2 is the present invention 236 The results of detecting the expression levels of related cytokines in the serum of mice after immunization with structural protein E+M, where: A is the expression level of TNF-α in mouse serum; B is the expression level of IL-4 in mouse serum; C is the expression level of IL-10 in mouse serum;

[0031] Figure 10 NSP2 is the present invention 236 The results of cytokine expression levels in splenic lymphocytes after immunization of mice with structural protein E+M, where: A represents the expression level of IL-4 mRNA; B represents the expression level of IL-6 mRNA; C represents the expression level of IL-10 mRNA; and D represents the expression level of TNF-α mRNA.

[0032] Figure 11 NSP2 is the present invention 236 The results of cellular immunity detection after immunization of mice with structural protein E+M are shown in the following figures: A is the result of ELISpot assay; B is the expression level of IFN-γ in splenic lymphocytes of mice 28 days after immunization; C is the secretion level of IFN-γ in splenic lymphocytes of mice 42 days after immunization; D is the expression of IFN-γ in serum; E is the proliferative activity of splenic lymphocytes of mice 28 days after immunization; and F is the proliferative activity of splenic lymphocytes of mice 42 days after immunization.

[0033] Figure 12 NSP2 is the present invention 236 Results of humoral immunity levels in mice after co-immunization with structural protein E+M, where: A represents the level of specific antibody IgG; B represents the level of specific antibody IgG1; C represents the level of specific antibody IgG2a; D represents the level of the co-immunized group (NSP2). 236 Analysis of the results of immune bias induced by E+M);

[0034] Figure 13 NSP2 is the present invention 236 Results of neutralizing antibody levels in mice after co-immunization with structural protein E+M;

[0035] Figure 14 NSP2 is the present invention 236Immunohistochemical results after immunizing mice with the structural protein E+M, where: A is the immunohistochemical result of mouse spleen (20×); B is CD3. + Percentage of T-cell positive area; C represents CD4. + The percentage of T-cell positive area; D represents CD8. + Percentage of T-cell positive area; E CD4 + T cells and CD8 + T cells relative to CD3 + Changes in the ratio of T cells. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0037] Materials and reagents:

[0038] PRRSV strain XJSW-2021 (GenBank accession number OR247780.1), Marc-145 cells, and PRRSV antibody-positive serum were preserved in our laboratory. Porcine reproductive and respiratory syndrome inactivated vaccine (CH-1a strain) was purchased from Zhejiang Meibaolong Biotechnology Co., Ltd. Six-week-old female BALB / c mice were purchased from the Henan Provincial Experimental Animal Center. Animal experiments were approved by the Bioethics Committee of Shihezi University (Approval No.: A2023-246).

[0039] Antibodies IgG (catalog number: 80015-1-RR), IgG1 (catalog number: 66360-1-Ig), IgG2a (catalog number: 98600-1-MR), His tag (catalog number: 66005-1-Ig), CD3 (catalog number: 60181-1-Ig), CD4 (catalog number: 67786-1-Ig), CD8 (catalog number: 66868-1-Ig), and goat anti-mouse IgG HRP (catalog number: SA00013-1) were purchased from Proteintech Biotechnology Co., Ltd. Goat anti-pig IgG HRP (catalog number: QYL-030636) was purchased from Qiyi Biotechnology Co., Ltd. The lymphocyte CCK8 assay kit (catalog number: C0037) was purchased from Beyotime Biotechnology Co., Ltd. Mouse IL-4 (Catalog No.: JL20266), IL-10 (Catalog No.: JLW20242), and TNF-α (Catalog No.: JL10484) ELISA kits were purchased from Jianglai Biotechnology Co., Ltd. Mouse spleen lymphocyte isolation kit (Catalog No.: LTS1092PK) was purchased from Tianjin Haoyang Biotechnology Co., Ltd. Mouse ELISA kit for IFN-γ (Catalog No.: 3321-4AST-10) was purchased from Mabtech.

[0040] Table 1 Sequence Information Table

[0041]

[0042] Example 1: Screening of subunit vaccine antigens

[0043] This application focuses on the NSP1-8 non-structural protein encoded by the ORF1a reading frame of the PRRSV NADC30 strain to systematically evaluate its immunogenic potential as a vaccine candidate antigen.

[0044] First, the physicochemical properties of the target protein were analyzed using Expasy-ProtParam (https: / / web.expasy.org / protparam / ), and its transmembrane domains were predicted using TMHMM (https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ) to exclude transmembrane regions that might affect antigen expression. For cellular immune epitope prediction, for cytotoxic T lymphocyte (CTL) epitopes, the IEDB MHC I prediction tool (https: / / www.iedb.org / ) was used to screen peptides with a percentile rank less than 0.5 as potential CTL epitopes based on the binding affinity of MHC molecules to antigen peptides. Simultaneously, the IEDB MHC-II prediction module was used for joint analysis of helper T lymphocyte (HTL) epitopes to comprehensively assess the antigen's potential for cellular immune response. In the prediction of humoral immune epitopes, three online tools, ABCpred (http: / / www.imtech.res.in / raghava / abcpred), SVMTriP (http: / / sysbio.unl.edu / SVMTriP / prediction.php) and BCpreds (http: / / ailab.cs.iastate.edu / bcpreds / predict.html), were used to systematically identify linear B-cell epitopes in target antigens.

[0045] By comprehensively comparing the prediction results of various tools, overlapping epitopes that were identified in all three methods and scored highly were selected as candidate B-cell epitopes. Through the above multi-step screening and comprehensive analysis, a fragment of the non-structural protein NSP2 (NSP2) was finally identified. 236 Its amino acid sequence (as shown in SEQ ID NO:1) exhibits high immunogenicity, containing potential CTL, HTL, and B cell epitopes. Based on bioinformatics analysis, NSP2 is predicted to... 236 The immunogenicity of NSP2 is superior to other fragments or the full-length NSP2 (Table 2). 236 The fragment contains a key antigenic epitope that can be efficiently processed, presented, and strongly activate the host's adaptive immune response. In other fragments or the full-length protein, this epitope may be unable to function effectively due to structural obfuscation, low processing efficiency, or the presence of immunosuppressive regions. These results indicate that NSP2... 236 It has potential for further development as a candidate antigen for PRRSV subunit vaccines.

[0046] Table 2. Antigen index analysis of different proteins

[0047]

[0048] Example 2: Subunit vaccine antigen expression and purification

[0049] 1. NSP2 236 Construction and enzyme digestion identification of recombinant plasmids

[0050] For NSP2 236 The gene sequence (nucleotide sequence shown in SEQ ID NO:2) was codon optimized and sent to Beijing Qingke Biotechnology Co., Ltd. for gene synthesis. The gene was then ligated into the pET-28a expression vector, and the recombinant plasmid was identified by double enzyme digestion. The recombinant plasmid digestion system is shown in Table 3, and the reaction conditions were 37℃ for 1 h.

[0051] Table 3 Double enzyme digestion system

[0052]

[0053] result NSP2 236 After enzyme digestion, the recombinant plasmid yielded two bands, measuring 5369 bp and 720 bp, which matched the size of the target band. Figure 1 (A) The bands that were correctly identified by enzyme digestion were recovered by gel extraction and sent for sequencing. The sequencing results showed a 100% match with the target gene, indicating that the correct recombinant plasmid was constructed.

[0054] 2. NSP2 236 Transformation of recombinant plasmids

[0055] BL21(DE3) competent cells were prepared and stored at -80℃. 10 μL of correctly identified recombinant plasmid was added to the BL21(DE3) competent cells, mixed, and incubated on ice for 30 min. Simultaneously, a water bath was preheated to 42℃. After the ice bath, the cells were quickly placed in the water bath for a 90-second heat shock, followed by an ice bath for 3-5 min. Then, 800 μL of antibiotic-free LB liquid medium was added, and the cells were incubated at 37℃ and 180 rpm with shaking for 60 min. After centrifugation at 8000 rpm for 2 min, the supernatant was discarded. The remaining approximately 100 μL of bacterial culture was mixed and precipitated. The transformation product was then spread onto LB solid medium containing kanamycin sulfate (50 μg / mL) and cultured overnight at 37℃. The next day, single colonies were picked and cultured on LB liquid medium containing kanamycin sulfate with shaking. The cultured bacterial culture was stored in glycerol tubes at a 1:1 ratio of bacterial culture to 50% glycerol and frozen at -20℃.

[0056] 3. NSP2 236 prokaryotic expression

[0057] Save NSP2 236 20 μL of glycerol bacteria were inoculated into two bottles of 20 mL liquid LB medium containing kanamycin sulfate. One bottle was incubated at 37℃ and 170 rpm on a shaker until the bacterial culture became turbid. At this point, 1 mL of the bacterial culture was collected into an EP tube and centrifuged at 12000 rpm for 2 min to collect the bacterial precipitate. Then, ITPG inducer was added at a volume ratio of 1:1000, and 1 mL of bacterial precipitate was collected after induction at 37℃ and 170 rpm for 2 h, 4 h, 6 h, and 8 h. The other bottle was incubated at 16℃ and 170 rpm on a shaker for 16 h, and 1 mL of bacterial precipitate was collected. The bacterial precipitates from different induction conditions and at different times were added to 80 μL of distilled water, followed by 20 μL of protein loading buffer (5×SDS-PAGE Loding Buffer) and mixed well. All the samples were then placed in a 100℃ metal bath for 10 min.

[0058] result Protein expression levels under different induction conditions were compared by SDS-PAGE electrophoresis. The results showed that after induction at 37℃ for 6 hours, the recombinant protein NSP2... 236 The highest expression level ( Figure 1 (B) This condition is determined to be the optimal induction parameter.

[0059] 4. NSP2 236 Purification

[0060] NSP2 induction at 37°C 236Six hours after protein expression, the bacteria were centrifuged at 8000 rpm for 20 minutes. The bacterial pellet was resuspended in PBS, sonicated, and then centrifuged at 12000 rpm for 30 minutes at 4°C. The supernatant was collected. The collected fragmented pellet was dissolved in 30 mL of 8 M urea and incubated overnight on an ice bath in a horizontal shaker. 25 mL of filtered deionized water and 8 mL of inclusion body solution A were added to the nickel column to equilibrate the strain. The dissolved protein was centrifuged at 12000 rpm for 1 h, and the supernatant was collected. The filtered supernatant was added to the column to collect the flow-through. 40 mL of filtered inclusion body solution A was used to elute impurities, followed by 15 mL of inclusion body solution B to elute the target protein. The eluent was collected, and the column was washed with 25 mL of filtered inclusion body solution A and deionized water. 15 mL of 20% ethanol was added to seal the column, and it was stored at 4°C. The collected target protein eluate was identified by SDS and then placed in a dialysis bag. The dialysis bag was then subjected to gradient dialysis in urea solutions of different concentrations: 6 M, 4 M, 2 M, and 1 M urea solutions for 6-8 h, followed by buffering in PBS buffer for 6 h. The dialyzed protein solution was collected, and the concentration of the purified protein was determined using a commercially available BCA kit. If the concentration was low, sucrose was used to concentrate the protein to a final concentration of 1-2 mg / mL, and the solution was stored at -20°C.

[0061] result SDS-PAGE showed that high-purity recombinant protein was obtained after nickel column purification. Figure 1 (C)

[0062] 5. Western Blotting Identification

[0063] The purified proteins were subjected to SDS-PAGE electrophoresis. After electrophoresis, the gel was cut according to the protein size, and the cut gel was immersed in a petri dish containing transfer buffer. PVDF membranes were cut to the appropriate size according to the gel strips, and the PVDF membranes were activated with methanol for 5 min. The sponge, filter paper, PVDF membrane, gel, filter paper, and sponge were placed sequentially in the transfer apparatus according to the positive and negative electrodes, and transfer was performed for 20 min. After transfer, the membrane was blocked overnight at 4°C for 4 h in 5% skim milk blocking buffer.

[0064] Primary antibody incubation: Incubate primary antibody diluted 1×TBST (1:5000) on a shaker at room temperature for 2 hours, followed by TBST washing 5 times for 10 minutes each time. (His tag detection: mouse anti-His tag monoclonal antibody; PRRS antigen detection: PRRS-positive porcine serum)

[0065] Secondary antibody incubation: Incubate the secondary antibody diluted 1×TBST (1:10000) at room temperature for 2 hours, followed by washing. (His tag detection: goat anti-mouse IgG-HRP; PRRS antigen detection: goat anti-pig IgG-HRP)

[0066] In a darkroom, ECL developer is prepared according to the specified ratio. After the washed PVDF membrane is slightly dried, it is gently placed into a chemiluminescence analyzer. ECL developer is then added to the membrane for development, and the membrane is photographed and analyzed.

[0067] result ( Figure 1 (D and E): The recombinant protein can specifically bind to His-tagged antibodies and PRRS-positive serum, with a band size of approximately 35 kDa, indicating that the recombinant protein NSP2... 236 It can not only be correctly expressed in vitro, but also has good immunogenicity.

[0068] Example 3 NSP2 236 Immunogenicity analysis

[0069] NSP2 236 (1 mg / mL) emulsified with Freund's adjuvant (catalog number: F5881 / 344291, Sigma) at a ratio of 1:1 (v / v). For the first immunization, use Freund's complete adjuvant; for the second immunization, use Freund's incomplete adjuvant. Refer to Table 4 for the specific immunization schedule. Figure 2 The immunization procedure shown in Figure A was performed. The immunization method was multiple subcutaneous injections into the back of mice. Before immunization, blood was collected from the tail of the mice as a negative control. After immunization, blood was collected from the tail every 7 days.

[0070] All immunization-related time points mentioned in the text are calculated from the day of the first immunization (denoted as 0 d). For example, "7 days after immunization" means the 7th day after the first immunization.

[0071] Table 4. Mouse Immunization Schedule

[0072]

[0073] Note: The control group Vaccines in Group 3 is the CH1a strain inactivated vaccine (i.e., the commercial vaccine group).

[0074] 1. Safety evaluation

[0075] Record the changes in mouse weight, mental state, and diet within 7 days after the first and second immunizations, and observe whether the mice exhibit any abnormal behavior after immunization.

[0076] result Mice within 7 days after the first immunization ( Figure 2 (B) and within 7 days after the second exemption ( Figure 2 In the C group, no weight loss was observed, and the weight remained stable throughout the immunization period. Furthermore, the mice exhibited good mental and dietary conditions and no abnormal behavior after immunization.

[0077] 2. HE staining and immunohistochemistry of spleen tissue sections

[0078] method Spleens were collected from mice at 42 days of age, immersed in 4% formaldehyde tissue fixation solution, and sent to Benroco Biotechnology Co., Ltd. for HE staining and immunohistochemistry. ImageJ software was used to quantify the immunohistochemical results of the mouse spleens.

[0079] Results of HE staining of spleen tissue sections Spleens were collected from 42-day-old mice. No gross changes in color, appearance, or size were observed in the spleens of the experimental groups during dissection. Histological sections were prepared and stained with hematoxylin and eosin (HE). The spleen tissue structure of all experimental groups was normal, with clear splenic corpuscles, visible central arteries, and no significant changes in the size or number of splenic nodules. The boundary between the white and red pulp was clear, and no obvious necrosis or inflammatory cell infiltration was observed. Figure 2 (D).

[0080] Immunohistochemical results CD3 + An increased T cell count indicates enhanced T cell immune function in the body; compared with the PBS control group, recombinant protein NSP2... 236 Immunizing mice can significantly upregulate CD3 levels in spleen tissue. + The percentage of positive T cells was significantly higher in the T cell group than in the vaccine group (P < 0.05). Figure 7 (A, B) CD4 + T cells are a subset of lymphocytes produced by the thymus and play an important role in cellular immune responses. NSP2 236 Group CD4 + The percentage of positive T cells was significantly higher in the PBS control group and the vaccine group than in the PBS control group (P < 0.05). Furthermore, the recombinant protein NSP2... 236 CD8 of the group + The percentage of T-cell positive area was significantly higher in the vaccine group than in the vaccine group. Figure 7 (A, D). Regarding CD4 + T cells and CD8 + Further analysis of the T cell percentage revealed CD4 + The proportion of T cells was higher than that of CD8. + T cells ( Figure 7 (E), namely, recombinant protein immunization of mice to stimulate the body's T lymphocytes to transform into CD4+. + T cell differentiation, while CD4 + The main function of T cells is to enhance the anti-infection effect mediated by phagocytes and the humoral immune response mediated by B lymphocytes. This further indicates that immunization with recombinant proteins can induce humoral immunity in the body.

[0081] 3. Quantitative PCR detection of cytokine mRNA expression levels

[0082] method Three mice were randomly selected at 28 days and 42 days of age and sacrificed. The spleens were aseptically dissected and isolated in a clean bench. Splenic lymphocytes were isolated according to the instructions of the lymphocyte isolation kit. The isolated splenic lymphocytes were seeded onto 24-well cell culture plates. Antigen-stimulated and unstimulated control groups were set up. After antigen stimulation, the 24-well plates were incubated for 24 h in a cell culture incubator before collection. RNA was extracted according to the instructions of the RNA extraction kit. After concentration measurement, the RNA was reverse transcribed into cDNA. The cDNA was used as a template for quantitative real-time PCR to detect the expression levels of various cytokine mRNAs. The primer sequences for each cytokine are shown in Table 1 (as shown in SEQ ID NO:3-10). The reaction conditions were 95℃ for 30 s, 95℃ for 5 s, 58℃ for 10 s, and 72℃ for 15 s, for 35 cycles. -ΔΔCt The method is used for relative quantitative analysis.

[0083] result At 28 and 42 days post-immunization, there were no significant differences in the expression levels of IL-4 and IL-10 mRNA between the experimental group and the PBS control group (P > 0.05). Figure 3 (A, C). On day 28, compared to the PBS control group, recombinant protein NSP2... 236 Immunization of mice significantly upregulated IL-6 mRNA expression (P < 0.001), and the expression level of IL-6 mRNA was significantly higher than that in the vaccine group; at 42 days, the recombinant protein NSP2... 236 The expression of IL-6 mRNA in immunized mice was significantly higher than that in the vaccine group (P < 0.05). Figure 3 (B). At 28 and 42 days, the recombinant protein NSP2 236 The expression level of TNF-α mRNA was significantly upregulated in the group, and was significantly higher than that in the vaccine group (P < 0.01). Figure 3 (D), indicating the recombinant protein NSP2 236 It can significantly induce the production of immune cytokines in mice.

[0084] 4. ELISApot assay for IFN-γ secretion levels and CCK8 assay for lymphocyte proliferation activity (cellular immunity level detection)

[0085] method Remove the ELISpot plate from its sealed packaging and wash four times with sterile PBS (200 μL / well). Incubate the plate with 1640 cell culture medium containing 10% fetal bovine serum (200 μL / well) at room temperature for at least 30 min. Add lymphocytes (1 x 10^4 cells) to four wells. 6(10 μL of ConA (100 μg / mL) was added to one well as a positive control, and one well was left unstimulated as a negative control; two more wells were added with 10 μL of protein stimulant as the experimental group. IFN-γ expression was detected according to the kit instructions.)

[0086] Lymphocytes were arranged at a ratio of 1×10 per well. 5 Cells were seeded in 96-well plates, stimulated with antigen, and then cultured in a cell culture incubator. After adding CCK solution for 4 h, the absorbance at 450 nm was read using a microplate reader, and the stimulation index SI was calculated as follows: SI = (stimulator wells - non-stimulatory control wells) / (non-specific stimulator wells - non-stimulatory control wells).

[0087] result IFN-γ secretion in mouse splenic lymphocytes was measured using ELISApot on days 28 and 42 post-immunization to evaluate cellular immunity levels. Results showed that NSP2... 236 After immunizing mice with this group, the expression of IFN-γ in mouse splenic lymphocytes was significantly upregulated, and significantly higher than that in the commercial vaccine group. Figure 4 (A, B). Lymphocyte proliferation assay results showed that the recombinant protein NSP2 236 It significantly improved lymphocyte proliferation capacity, and was higher than that of the commercially available vaccine group ( Figure 4 (C) The above results indicate that NSP2 236 It can enhance the cellular immunity level in mice.

[0088] 5. Indirect ELISA detection of serum specific antibody levels (humoral immunity level detection)

[0089] method The checkerboard titration method was used to systematically screen mouse serum 28 days after immunization. Optimal reaction conditions, including the concentration of coating antigen, the dilution ratio of primary antibody, and the working concentration of secondary antibody, were determined through orthogonal experiments. Indirect ELISA was then performed according to the selected optimal reaction conditions: the antigen was diluted with coating buffer and coated onto the ELISA plate overnight at 4°C; the plate was blocked with 5% skim milk powder (prepared with PBS) at 37°C for 2 hours; the test serum (primary antibody) was added and incubated at 37°C for 2 hours; after washing with PBST, the corresponding subtype-specific enzyme-labeled secondary antibody (e.g., HRP-goat anti-mouse IgG1 for IgG1, HRP-goat anti-mouse IgG2a for IgG2a) was added and incubated at 37°C for 1 hour; subsequently, TMB chromogenic solution was added and reacted in the dark for 15 minutes to terminate the reaction. The absorbance value was immediately read at 450 nm using an ELISA reader.

[0090] result Recombinant protein NSP2 236Following immunization of mice, the level of specific antibody IgG generally showed an upward trend, with a slight increase starting after day 14 and continuing until day 42. However, no significant changes in antibody levels were observed in the commercial vaccine group and the PBS group throughout the entire immunization period. Figure 5 (A). Detection results of specific antibody subtypes IgG1 and IgG2a indicate that NSP2 236 Group IgG1 began to rise on day 14 after the first immunization and continued to rise until day 42. Figure 5 IgG2a only began to rise after day 28 (B), while IgG2a only began to rise after day 28 (B). Figure 5 The levels of C (in the middle digit range) were higher than those in the commercial vaccine group. These results indicate that the recombinant protein can induce high levels of humoral immunity in mice.

[0091] Results of immune bias analysis To determine the recombinant protein NSP2 236 The immune bias induced in mice was further analyzed by examining the specific antibody subtypes in mouse serum. The ratio of the specific antibody subtypes IgG2a to IgG1 in mice was less than 1 (e.g., ...). Figure 5 As shown in D, 14-42 d), this illustrates NSP2. 236 The specific IgG produced in mice is biased towards subtype IgG1, and the immune type induced in mice is biased towards Th2 humoral immunity.

[0092] 6. Neutralization test to detect neutralizing antibody levels

[0093] method : Pre-determination of the TCID of the virus (PRRSV XJSW-2021 strain) 50 Large-scale culture of Marc-145 cells was conducted. Serum from 28-day and 42-day-old mice was heat-inactivated one day prior to culture at 56°C for 30 minutes. The cultured cells were then evenly seeded onto 96-well cell culture plates at a density of 8000 cells per well. The next day, the heat-inactivated serum was seeded onto 96-well U-shaped plates at a density of 2... 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 Perform serial dilutions, and add an equal volume of virus solution (200 TCID50) to the serially diluted serum. 50After incubating in a cell culture incubator for 2 hours, the serum-virus solution from a 96-well U-shaped plate was transferred to a 96-well cell culture plate and incubated with cells for 1 hour. The solution was then replaced with DMEM maintenance medium containing 1% fetal bovine serum. Cytopathic effects were observed for one week, and a positive result for neutralizing antibodies was defined as the ability to protect 50% of cells from viral infection. The titer of neutralizing antibodies (NA) was calculated using the Reed-Muench method.

[0094] result On days 28 and 42 after the first immunization, recombinant protein NSP2... 236 The levels of neutralizing antibodies induced in the immunized group and the commercial vaccine group were significantly higher than those in the PBS control group (p < 0.01). Based on log2 titer conversion, NSP2... 236 The neutralizing antibody titers in the recombinant NSP2 group at 28 and 42 days were approximately 1:107 and 1:99, respectively, while the titers in the commercial vaccine group at the same time points were approximately 1:39 and 1:62. This indicates that at both time points, the recombinant NSP2 protein... 236 The induced neutralizing antibody titers were all higher than those of commercially available vaccines. This result confirms that the recombinant protein NSP2... 236 It can induce a high level of neutralizing antibody response in mice, and its effect is superior to that of the commercially available vaccines used.

[0095] Example 4 NSP2 236 Combined immunization with structural protein E+M

[0096] The amino acid sequence and coding sequence of the PRRSV tandem structural protein E+M are shown in Table 1. Specifically, the tandem structural protein E+M is formed by covalently linking the PRRSV E protein (GenBank accession number: ACG60052.2, amino acids 1-14 and 33-200) and M protein (NCBI RefSeq accession number: NP_047412.1, amino acids 1-40 and 96-174) via a flexible linker peptide (GGGGS) after reasonable optimization. The designed sequence was codon-optimized, sent to Beijing Qingke Biotechnology Co., Ltd. for gene synthesis, and ligated into the pET-28a expression vector. Expression and purification were performed according to the method in Example 3. The purified protein and adjuvant were emulsified at a ratio of 1:1 (50 μg protein: 50 μL adjuvant), and then expressed according to Table 5. Figure 8 The mice were immunized using the A-immunization program. The immunization method involved subcutaneous injection at multiple sites on the back of the mice. Before immunization, blood was collected from the tails of the mice as a negative control. After immunization, blood was collected from the tails every 7 days.

[0097] Table 5. Immunization schedule for mice

[0098]

[0099] Note: The control vaccine in group 4 is the CH1a strain inactivated vaccine.

[0100] 1. Safety evaluation, HE staining and immunohistochemistry of spleen tissue sections

[0101] method The safety evaluation, HE staining of spleen tissue sections, and immunohistochemistry methods are the same as in Example 3.

[0102] result Compared with the PBS group, the immunized mice showed good mental and dietary status and no weight loss after immunization. Figure 8 (B and C) No abnormal behavior was observed throughout the immunization period.

[0103] HE staining of spleen tissue sections showed that the spleen tissue structure was normal in all experimental groups, the lymph node structure was clear, and the boundary between the white pulp and the red pulp was clear. Figure 8 These results (including those from the Chinese Center for Disease Control and Prevention) indicate that the combined immunization is safe and well-tolerated.

[0104] Immunohistochemical results showed that recombinant protein NSP2 236 Combined immunization with E+M significantly activated cellular immune responses; all recombinant protein immunization groups significantly increased the percentage of positive areas for CD3⁺ T cells (total T cells); combined immunization group (NSP2) 236 The effect of immunization with E+M was the strongest, significantly higher than that of immunization with E+M alone and immunization with NSP2 alone. 236 Group (P < 0.01) Figure 14 (A and B in the middle).

[0105] Recombinant protein NSP2 236 Both the group receiving the vaccine and the combined immunization group induced a strong CD4⁺ T cell response, with a significantly higher percentage of positive responses compared to the commercial vaccine group and the PBS group (P < 0.05). Figure 14 (A, C). Furthermore, the percentage of CD8⁺ T cells positive in all recombinant protein immunization groups was significantly higher than in the PBS group, and the combined immunization group was significantly more effective than the E+M group alone, while there was no significant difference between the commercial vaccine group and the PBS group (P < 0.05). Figure 14 (A and D in the original text). The overall proportion of CD4⁺ T cells was higher than that of CD8⁺ T cells, indicating that the immune response was biased towards CD4⁺ T cells. Figure 14 (E).

[0106] The results showed that NSP2 236 It can significantly enhance the adaptive immune response induced by E+M protein.

[0107] 2. ELISA kit was used to detect the expression of relevant cytokines in mouse serum.

[0108] method Serum was collected from mice on day 42, and the expression of IL-4, IL-10, TNF-α, and IFN-γ in the serum was detected according to the ELISA kit instructions.

[0109] result Recombinant protein NSP2 at 42 days 236 Group and combined immunization NSP2 236 The expression level of TNF-α in the serum of the combined immunization group and the E+M group was significantly higher than that in the vaccine group and the PBS control group. Furthermore, the expression level of TNF-α in the serum of the combined immunization group was significantly higher than that in the serum of the E+M group immunized alone. Figure 9 (A). At 42 days, the expression level of IL-4 in the immunized group was significantly higher than that in the PBS group, and the expression level was also significantly higher in the combined immunization group with NSP2. 236 The expression level of IL-4 in the serum of the +E+M group was significantly higher than that in other experimental groups. Figure 9 (B) At 42 days, the expression level of IL-10 in the serum of the E+M group immunized alone was significantly higher than that in the vaccine group and the PBS control group, while the expression level of IL-10 in the combined immunization NSP2 group was significantly higher. 236 The +E+M group showed no significant difference from the vaccine group and the PBS control group (P>0.05). Figure 9 (C). These results indicate that combined immunization (NSP2) 236 E+M protein combination therapy effectively promotes the release of immune-activating factors such as TNF-α without significantly upregulating the immunosuppressive factor IL-10. Compared with E+M protein alone, combined immunization is more beneficial for maintaining immune activation.

[0110] 3. Quantitative PCR detection of cytokine mRNA expression levels

[0111] method Same as Example 3, "Quantitative Detection of Cytokine mRNA Levels".

[0112] result :like Figure 10 As shown in Figure A, at 28 days post-immunization, compared with the PBS control group, the IL-4 mRNA level was significantly upregulated in the E+M group and the commercial vaccine group immunized alone, while the level was significantly upregulated in the NSP2 group immunized alone. 236 The expression levels in the control group and the combined immunization group were not significantly different from those in the PBS control group. By day 42, the IL-4 mRNA expression levels in all experimental groups were not significantly different from those in the PBS control group.

[0113] like Figure 10As shown in Figure B, at both 28 and 42 days, the expression levels of IL-6 mRNA in all experimental groups were significantly higher than those in the PBS control group. Furthermore, at 42 days, the expression levels in all experimental groups were also significantly higher than those in the commercial vaccine group (P < 0.01).

[0114] like Figure 10 As shown in Figure C, only at 28 days post-immunization, the E+M group alone showed a significant upregulation of IL-10 mRNA expression; the expression levels in the other experimental groups at different time points were not significantly different from those in the PBS control group. Figure 10 As shown in Figure D, at 28 and 42 days, the expression levels of TNF-α mRNA in all experimental groups were significantly higher than those in the commercial vaccine group and the PBS control group (P < 0.01). Among these, immunization with NSP2 alone... 236 The expression levels in the group and the combined immunization group were significantly higher than those in the commercial vaccine group (P < 0.05).

[0115] 4. ELISApot assay for IFN-γ secretion levels and CCK8 assay for lymphocyte proliferation activity (cellular immunity level detection)

[0116] method : Same as the corresponding part in Example 3.

[0117] result On day 28, the IFN-γ expression levels in all immunization groups were significantly higher than those in the PBS control group, with the combined immunization with NSP2 showing the highest levels. 236 The IFN-γ expression level in the +E+M group was significantly higher than that in the immunization-only group and the commercial vaccine group (P<0.01). Figure 11 (A and B). At 42 days, combined immunization with NSP2. 236 The IFN-γ expression level in the +E+M group was significantly higher than that of NSP2 alone. 236 Group and E+M group ( Figure 11 (C). In addition, NSP2 236 Immunizing mice in the +E+M group significantly upregulated serum IFN-γ expression, and the serum IFN-γ expression level in the combined immunization group was significantly higher than that in the NSP2 immunization alone group. 236 The expression level of IFN-γ in serum of the group or the E+M group alone ( Figure 11 (D).

[0118] The lymphocyte proliferation assay results showed that, at 28 days, NSP2 immunization alone... 236 Group, combined immunization with NSP2 236 The +E+M group and the vaccine group significantly improved the proliferation capacity of splenic lymphocytes in mice. Figure 11In the PBS group (E), at 42 days, there was no significant difference in lymphocyte proliferation capacity between the experimental groups and the vaccine and PBS groups. Figure 11 (F). The above results indicate that the recombinant protein NSP2 236 Combined immunization with E+M protein can effectively induce cellular immunity.

[0119] 5. Indirect ELISA detection of serum specific antibody levels (humoral immunity level detection)

[0120] method : Same as the corresponding part in Example 3.

[0121] result Regarding humoral immune responses ( Figure 12 (A), combined immunization (NSP2) 236 The E+M group induced high levels of specific IgG as early as 14 days after immunization, while the E+M group and NSP2 group immunized alone... 236 Significant levels of IgG were not produced until day 28; by day 42, the IgG levels in the combined immunization group remained higher than those in each individual immunization group. Analysis of antibody subtypes showed ( Figure 12 In experimental groups B and C, IgG1 levels showed a continuous upward trend from day 14, while IgG2a levels only began to rise slightly after day 28, reaching a significant level at day 42. To further clarify the type of immune response, we analyzed the ratio of IgG2a to IgG1 (…). Figure 12 (D). The results showed that the ratio in each experimental group was consistently less than 1, indicating that both single and combined immunization mainly stimulated a humoral immune response biased towards IgG1, i.e., a Th2-type immune bias.

[0122] 6. Neutralization test to detect neutralizing antibody levels

[0123] method : Same as the corresponding part in Example 3.

[0124] result :like Figure 13 28 days after immunization, the experimental groups (E+M, NSP2) showed... 236 The neutralizing antibody levels in both the combined immunization group and the commercially available vaccine group (Vaccines) were significantly higher than those in the PBS control group. The combined immunization group showed the most significant effect, with antibody levels significantly higher than the vaccine group. By 42 days post-immunization, antibody levels in the combined immunization group further increased and were significantly higher than in all other groups. Furthermore, antibody levels in both the vaccine group and the E+M group were also significantly higher than in the PBS group at this time point.

[0125] The neutralizing antibody titers in the combined immunization group were approximately 1:115 and 1:150 at 28 and 42 days, respectively, and were consistently significantly higher than those of NSP2 alone. 236 The NSP2 group also outperformed the E+M group, with the advantage becoming more pronounced at 42 days. This indicates that NSP2... 236 Combined immunization with E+M can continuously produce high levels of neutralizing antibodies in mice.

[0126] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A porcine reproductive and respiratory syndrome virus antigen, characterized in that, The amino acid sequence of the antigen is shown as SEQ ID NO:

1.

2. The antigen of claim 1, characterized in that, The nucleotide sequence of the gene encoding the antigen is shown as SEQ ID NO:

2.

3. Use of the antigen of claim 1 or 2 in the preparation of a product for preventing and treating porcine reproductive and respiratory syndrome.

4. An antigen combination, characterized by The antigen combination comprises the antigen of claim 1.

5. The antigen combination of claim 4, characterized in that, The antigen combination further comprises an antigen protein with an amino acid sequence shown as SEQ ID NO:

11.

6. Use of the antigen combination of claim 4 or 5 in the preparation of a product for preventing and treating porcine reproductive and respiratory syndrome.

7. A subunit vaccine of porcine reproductive and respiratory syndrome virus, characterized in that, The vaccine comprises the antigen of claim 1 and a vaccine adjuvant.

8. The vaccine of claim 7, characterized in that, The adjuvant is Freund's adjuvant.

9. The vaccine of claim 8, characterized in that, The volume ratio of the adjuvant to the antigen is 1:

1.

10. An expression vector, characterized by, The expression vector comprises a gene fragment with a nucleotide sequence shown as SEQ ID NO: 2.

Images