A PCV2-SVA bivalent vaccine

Abstract

The present invention belongs to the technical field of veterinary drug production, and provides a PCV2-SVA bivalent vaccine. The vaccine uses the PCV2 Cap protein with the nuclear localization signal removed and the SVA VP2 protein as antigens. The present invention efficiently expresses soluble PCV2 Cap protein and SVA VP2 protein through the baculovirus system. Due to the immune synergy, compared with commercial vaccines or inactivated vaccines, the PCV2-SVA bivalent vaccine prepared based on recombinant proteins can stimulate higher levels of humoral immunity and cellular immunity. The challenge protection test after immunization shows that the bivalent vaccine can effectively reduce the viremia and the virus content in organs. The bivalent genetic engineering vaccine against PCV2 and SVA developed based on the present invention has commercial development prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary drug production, and particularly relates to a bivalent vaccine with PCV2 Cap protein and SVA VP2 protein as antigens. Background Art

[0002] Disclosing the information of this background art aims to enhance the understanding of the overall background of the present invention, and it is not necessarily regarded as an admission or an indication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Porcine circovirus type 2 (PCV2) is a major pathogen in the global pig industry, especially causing immunosuppressive diseases such as porcine postweaning multisystemic wasting syndrome, among which the PCV2d strain is the most prevalent. At the same time, Senecavirus A (SVA) is a highly contagious virus, which is likely to cause clinical symptoms similar to foot-and-mouth disease and is often co-infected with PCV2.

[0004] Both PCV2 and SVA are important infectious diseases affecting the pig farming industry, causing great economic losses to pig farms and severely restricting the development of the pig farming industry. Currently, there is no commercialized vaccine for SVA in the domestic and international markets, and there is a lack of combined vaccine products that can effectively prevent and control these two viruses. Therefore, in the current situation of the lack of an effective commercial vaccine against SVA and a combined vaccine against these two viruses, there is an urgent need for the research and development of new vaccine technologies. Summary of the Invention

[0005] Aiming at the problems in the prior art, the present invention provides a bivalent vaccine with PCV2 Cap protein and SVA VP2 protein as antigens. Due to the immune synergy, the bivalent vaccine has better humoral immunity and cellular immunity effects than the single vaccine group.

[0006] To achieve the above object, the present invention adopts the following technical solutions.

[0007] A coding nucleic acid of SVA VP2 protein, whose nucleotide sequence is as shown in SEQ ID NO: 1. This nucleic acid can efficiently and solubly express SVA VP2 protein in the insect baculovirus expression system.

[0008] The present invention also provides a vector and a cell containing the above coding nucleic acid. The above vector and cell can be used to express SVA VP2 protein and prepare SVA vaccine. Preferably, the expression system is the insect baculovirus expression system.

[0009] The vector is preferably selected from pFastBac-Dual vector and bMON14272 vector.

[0010] The cells are preferably selected from DH10Bac competent cells, Sf 9 cells, Sf 21 cells or Hi-5 cells.

[0011] The present invention also provides a PCV2-SVA bivalent vaccine, which uses the PCV2 Cap recombinant protein (Cap ΔNLS) with the nuclear localization signal removed and the SVA VP2 recombinant protein as antigens.

[0012] The amino acid sequence of the Cap ΔNLS is shown as SEQ ID NO: 2; the amino acid sequence of the SVA VP2 recombinant protein is shown as SEQ ID NO: 3.

[0013] The present invention has the following advantages:

[0014] The present invention efficiently expresses soluble PCV2 Cap protein and SVA VP2 protein through the baculovirus system. Due to the immune synergy, compared with commercial vaccines or inactivated vaccines, the PCV2-SVA bivalent vaccine prepared based on recombinant proteins can stimulate higher levels of humoral immunity and cellular immunity. The challenge protection test after immunization shows that the bivalent vaccine can effectively reduce the viremia and the virus content in organs. The bivalent genetic engineering vaccine against PCV2 and SVA developed based on the present invention has commercial development prospects. Description of the Drawings

[0015] Figure 1 is the map of the pFastBac Dual vector;

[0016] Figure 2 is the electrophoresis pattern after double digestion of the recombinant plasmid with BamH I and Hind III. Among them, M is DL10000 Marker; 1-9 are respectively: pFBD-VP1, pFBD-VP2, pFBD-VP2-ZYH, pFBD-VP3, pFBD-2CapΔNLS, pFBD-2Cap, pFBD-1CapΔNLS, pFBD-1Cap, empty vector; 10 is the negative control;

[0017] Figure 3 is the nucleic acid electrophoresis pattern for verifying the recombinant bacmid. Among them, M is DL10000 Marker; in A, 1 is only the bacmid; 2-5 are respectively: rBacmid-VP1, rBacmid-VP3, rBacmid-VP2-ZYH and rBacmid-VP2; in B, 1-4 are respectively rBacmid-1Cap, rBacmid-1CapΔNLS, rBacmid-2Cap, rBacmid-2CapΔNLS; 5 is the negative control;

[0018] Figure 4Morphological diagrams of sf9 cells and normal sf9 cells after inoculation with baculovirus (20×). Among them, A: normal cells; B: infected with rBV-1Cap; C: infected with rBV-1CapΔNLS; D: infected with rBV-VP1; E: infected with rBV-VP2; F: infected with rBV-VP2-ZYH; G: infected with rBV-VP3; H: infected with wild-type baculovirus;

[0019] Figure 5 Western blot results of proteins in the culture supernatant of sf9 cells. Among them, in A, M is the protein Marker, and 1-5 are infected with rBV-VP1, rBV-VP2-ZYH, rBV-VP2, rBV-VP3, and blank plasmid respectively;

[0020] Figure 6 Levels of cytokines IL-4 and IFN-γ in the serum of mice after immunization;

[0021] Figure 7 Levels of PCV2 and SVA IgG antibodies in the serum of mice after immunization;

[0022] Figure 8 PCV2 virus load in the organs of mice 28 days after virus challenge;

[0023] Figure 9 SVA virus load in the serum of mice at different times after virus challenge. Detailed implementation manners

[0024] The present invention will be further described below in conjunction with examples and drawings, but the present invention is not limited by the following examples.

[0025] Example 1 Preparation of recombinant protein

[0026] 1. Preparation of recombinant plasmid

[0027] The amino acid sequence information of VP1, VP2, and VP3 was obtained according to SVA (GenBank accession number: MZ818785.1). According to the OptimumGene method, codon-optimized SVA VP1 (792 bp, SEQ ID NO: 4), VP2 (852 bp, SEQ ID NO: 5), and VP3 (717 bp, SEQ ID NO: 6) were obtained. The start and stop codons were supplemented, and a 6×His tag (SEQ ID NO: 9) and BamH I restriction site were introduced at the 5' end, and a Hind III restriction site was introduced at the 3' end for commercial synthesis to obtain the target genes. In addition, the VP2 gene was codon-optimized by itself, denoted as SVA VP2-ZYH (852 bp, SEQ ID NO: 1), and a His tag (SEQ ID NO: 10) and the same restriction sites were added for commercial synthesis. The four gene coding sequences optimized by the above different methods were respectively ligated to the downstream of the pH promoter in the pFastBac-Dual vector, and green fluorescent protein (EGFP) was ligated to the downstream of the p10 promoter in the pFastBac-Dual vector. The recombinant plasmids were named pFBD-VP1, pFBD-VP2, pFBD-VP2-ZYH, and pFBD-VP3 respectively.

[0028] According to the nucleotide sequence of PCV2 Cap protein (GenBank accession number: JQ653449), after removing the nuclear localization signal (NLS), the base sequence of Cap protein was codon-optimized according to the codon preference of Sf9 cells. The optimized PCV2 Cap protein coding sequence (699 bp, SEQ ID NO: 7) or PCV2 CapΔNLS protein coding sequence (579 bp, SEQ ID NO: 8) was added with a 6×His tag (SEQ ID NO: 10) and GP67 signal peptide (SEQ ID NO: 11) at the 3' end, and the start and stop codons and restriction sites were supplemented for commercial synthesis to obtain the target genes. The target genes were respectively ligated downstream of the pH promoter in the pFastBac-Dual vector and named pFBD-1Cap and pFBD-1CapΔNLS. Specific primers were designed according to the above recombinant plasmids to amplify the target genes containing the Cap gene or CapΔNLS gene with Xho I and Kpn I restriction sites at both ends, and they were ligated to the downstream of the p10 promoter in the pFBD-1Cap and pFBD-1CapΔNLS vectors to form double-copy recombinant vectors pFBD-2Cap and pFBD-2CapΔNLS.

[0029] The constructed recombinant plasmids pFBD-VP1, pFBD-VP2, pFBD-VP2-ZYH, pFBD-VP3, pFBD-2CapΔNLS, pFBD-2Cap, pFBD-1CapΔNLS, and pFBD-1Cap were identified by double digestion, and the electrophoresis results are as Figure 2 shown: After double digestion, each recombinant vector had two bands, the empty vector had only one band, and the blank control had no band. According to the theoretical design, the two bands after double digestion of pFBD-VP1 were 5958 bp and 879 bp respectively; the two bands after double digestion of pFBD-VP2 and pFBD-VP2-ZYH were 5958 bp and 939 bp respectively; the two bands after double digestion of pFBD-VP3 were 5958 bp and 804 bp respectively; the two bands after double digestion of pFBD-2CapΔNLS were 5859 bp and 621 bp respectively; the two bands after double digestion of pFBD-2Cap were 5979 bp and 741 bp respectively; the two bands after double digestion of pFBD-1CapΔNLS were 5238 bp and 621 bp respectively; the two bands after double digestion of pFBD-1Cap were 5238 bp and 741 bp respectively, all consistent with the results in the figure. Combining with the sequencing results, it indicated that the recombinant vectors were constructed correctly.

[0030] 2. Preparation of recombinant virus

[0031] The obtained PCV2 Cap and each SVA recombinant positive plasmid were transferred into DH10Bac competent cells by heat shock method, screened and extracted bacmids for identification, and the positive bacmids were named rBacmid-1Cap, rBacmid-1CapΔNLS, rBacmid-2Cap, rBacmid-2CapΔNLS, rBacmid-VP1, rBacmid-VP2, rBacmid-VP2-ZYH, and rBacmid-VP3 respectively. Identification was carried out with specific primers, and the results are as Figure 3 shown: The amplified target bands were consistent with the expected results rBacmid-1Cap (3301 bp), rBacmid-1CapΔNLS (3181 bp), rBacmid-2Cap (4042 bp), rBacmid-2CapΔNLS (3802 bp), rBacmid-VP1 (3439 bp), rBacmid-VP2 (3499 bp), rBacmid-VP2-ZYH (3499 bp), and rBacmid-VP3 (3364 bp).

[0032] The above recombinant bacmids were transfected with Cellfectin TMThe II reagent was transfected into Sf9 cells by the liposome method respectively, and the cells were cultured for 4 - 7 d until cytopathic effect (CPE) appeared. The supernatant of the cells with obvious CPE was harvested. After centrifugation at 1000 g for 5 min, the supernatant was taken and designated as the venom of the P1 generation. The harvested virus solutions were labeled as rBV-1Cap, rBV-1CapΔNLS, rBV-2Cap, rBV-2CapΔNLS, rBV-VP1, rBV-VP2, rBV-VP2-ZYH, and rBV-VP3.

[0033] The virus solution of the P1 generation was added to the adherent Sf9 cells in the logarithmic growth phase. After obvious CPE occurred, the cell supernatant was harvested. After centrifugation at 1000 g for 5 min, it was designated as the venom of the P2 generation, and was stored in the dark at 4℃ in the short term and in a -80℃ refrigerator in the long term. And the passage was carried out according to the above method.

[0034] After Sf9 cells were infected with recombinant baculoviruses rBV-1Cap (B), rBV-1CapΔNLS (C), rBV-VP1 (D), rBV-VP2 (E), rBV-VP2-ZYH (F), rBV-VP3 (G) and wild-type baculovirus empty vector (H) for 4 d, under the microscope ( Figure 4 ), it could be seen that the normal Sf9 cells were regular round or epithelial-like in shape under microscopic observation and had the ability of adherent growth and suspension growth. Compared with the normal uninfected Sf9 cells (A), the cells infected with recombinant baculoviruses showed an increase in volume. As time went by, most cells showed cytopathic phenomena such as an increase in cell volume, an increase in nuclear volume, and cell growth arrest.

[0035] 3. Expression of recombinant protein

[0036] Vectors rBacmid-1Cap, rBacmid-1CapΔNLS, rBacmid-2Cap, rBacmid-2CapΔNLS, rBacmid-VP1, rBacmid-VP2, rBacmid-VP2-ZYH, and rBacmid-VP3 were transfected into Sf9 cells respectively. After blind passage to the second generation, the cell supernatant was centrifuged and loading buffer was added. After SDS-PAGE electrophoresis, His-tag was used as the primary antibody for Western blot detection. The results were as Figure 5As shown, for the Cap protein, the expression level of the vector protein containing the target gene at both ends after the promoter is higher than that of the vector containing only one segment of the target gene, and the expression level of the truncated PCV2 Cap protein is higher than that of the complete Cap protein. Therefore, rBV-2CapΔNLS was selected for subsequent experiments to continue to highly express the PCV2 Cap protein. For each capsid surface protein of SVA, the expression levels of VP1 and VP2 are both relatively high. The expression level of the VP2-ZYH protein is higher than that of VP2. Since the nucleotides of VP1 are prone to mutation, resulting in antigenic variation, therefore, the efficient expression of VP2-ZYH was selected.

[0037] The recombinant baculovirus was continuously passaged to the P4 generation, and the virus titer of the obtained P4 generation recombinant baculovirus solution was accurately measured. The median tissue culture infective dose (TCID 50 50) of the virus was calculated using the Reed-Muench method, and then the plaque forming units (PFU) titer of the virus was deduced. The titers of rBV-2CapΔNLS and rBV-VP2-ZYH were 10 5.4 TCID 50 50 / mL and 10 6.6 TCID 50 50 / mL, respectively.

[0038] The virus solutions of rBV-2CapΔNLS and rBV-VP2-ZYH were inoculated into Hi-5 cells (2×10 6 cells / mL) at an MOI of 0.1, and the supernatant was harvested on the 4th day. The protein was purified using a Ni column. After protein purification, the expression level of the CapΔNLS protein in each mL of suspended Hi-5 cells was 0.125 mg; the expression level of the VP2 protein was 0.475 mg, which was a high-level expression compared with other similar domestic studies.

[0039] Example 2 Preparation of Bivalent Vaccine

[0040] The purified CapΔNLS protein and VP2 protein prepared in Example 1 were respectively mixed with JLC-3 water-soluble carbomer adjuvant at a ratio of 1:1 (m / m) and stirred at 300 rpm for 10 - 20 min; subunit vaccines were prepared according to the following contents:

[0041] (1) rBV-Cap monovalent vaccine: The content of Cap protein is 0.25 mg / mL;

[0042] (2) rBV-VP2 monovalent vaccine: The content of VP2 protein is 0.25 mg / mL;

[0043] (3) 25 μg group of rBV-Cap-VP2 combined vaccine: The content of Cap protein is 0.25 mg / mL, and the content of VP2 protein is 0.125 mg / mL

[0044] (4) 50 μg group of rBV-Cap-VP2 combined vaccine: The content of Cap protein is 0.25 mg / mL, and the content of VP2 protein is 0.25 mg / mL;

[0045] (5) 100 μg group of rBV-Cap-VP2 combined vaccine: The content of Cap protein is 0.25 mg / mL, and the content of VP2 protein is 0.5 mg / mL;

[0046] (6) SVA inactivated vaccine: Add β-propiolactone to the SVA-CH-SDGT-2017 strain (titer 10 9 TCID 50 / mL) at a ratio of 1 / 4000 (V / V) of the total volume of the virus solution, mix well, and inactivate at 2 - 8 °C for 24 h, stirring several times during this period; then hydrolyze the inactivated virus solution in a 37 °C water bath for 2 h; then take 2 mL of the inactivated virus and mix it with JLC-3 water-soluble carbomer adjuvant at a ratio of 1:1 (m / m), and stir at 300 rpm for 10 - 20 min;

[0047] Since the related vaccines of PCV2 virus have been widely studied in terms of immunogenicity, the antigen amount of the Cap protein in the combined vaccine is uniformly 0.25 mg / mL.

[0048] After the above vaccines are prepared, the quality of the vaccines is inspected in terms of appearance, physicochemical properties, stability, sterility, etc. Each prepared vaccine is a white or off-white homogeneous suspension liquid without insoluble phenomenon; the pH value ranges from 6.6 to 7.0; each vaccine is placed in a 1.5 mL EP tube and centrifuged at 3000 rmp for 15 min, and no solution stratification or precipitation is observed, proving that the vaccine has good stability; the prepared vaccines are respectively inoculated on TSA agar medium, and then after culturing at 37 °C for 48 h, no microbial growth is detected on the plate, indicating that the vaccine has passed the sterility inspection standard.

[0049] Application Example 1 Immunological Evaluation of the Vaccine on Mice

[0050] Take 80 healthy SPF-grade BALB / c male mice at 6 - 8 weeks old, randomly divide them into 8 groups, and immunize the mice with the vaccine prepared in Example 2 by intraperitoneal injection and gavage. The specific immunization protocol is shown in Table 1:

[0051] Table 1 Immunization Protocol

[0052]

[0053] At 0 d, 7 d, 14 d, 21 d, and 28 d after immunization respectively, the mice were anesthetized with ether in a fume hood, and then blood was collected from the retroorbital veins of the mice. The serum was separated by centrifugation at 4000 rpm for 15 min and stored at -20 °C for subsequent detection. The indirect ELISA method was used to detect cytokines IL-4 and IFN-γ and the level of specific antibody IgG in the serum of mice after immunization.

[0054] Twenty-eight days after immunization, the immunized mice were challenged with the virus. The titer of the PCV2 strain SD JQ653449 was 10 5.5 TCID 50 / mL; the titer of the SVA strain SVA-CH-SDGT-2017 was 10 9 TCID 50 / mL. The experimental protocol is shown in Table 2:

[0055] Table 2 Challenge protocol

[0056]

[0057] Fourteen days after challenge, the heart, lung, liver, spleen, kidney, brain, small intestine and other tissues and organs of all mice challenged with SVA were collected. Twenty-eight days after challenge, the heart, lung, liver, spleen, kidney and other tissues and organs of all mice challenged with PCV2 were collected. The viral load and histopathological changes were analyzed.

[0058] The levels of cytokines IL-4 and IFN-γ in the serum of mice at 7, 14, 21, and 28 d after immunization were as Figure 6 shown: Over time, except for PBS (blank control group), the level of IL-4 showed an overall trend of first increasing and then decreasing and was significantly different from the PBS group. The cytokine reached the highest level at 21 d. At 21 d after vaccination, the levels of cytokine IL-4 in the rBV-Cap-VP2 bivalent vaccine (25 μg, 50 μg, 100 μg) were higher than those in the rBV-Cap and rBV-VP2 monovalent vaccines. Among them, the 100 μg group of the rBV-Cap-VP2 bivalent vaccine induced the highest level of IL-4 cytokine, and the difference was extremely significant higher than that of the SVA inactivated vaccine group (P < 0.001) and the PCV2 commercial vaccine (P < 0.01);

[0059] With the increase in the number of days of vaccination, except for the PBS group, the IFN-γ levels in all other groups showed a trend of first increasing and then decreasing, and there were significant differences compared with the PBS group (P < 0.01). By the 21st day, the expression level of the cytokine IL-4 reached its peak, and the IFN-γ levels in all vaccinated groups were extremely significantly higher than those in the PBS group (P < 0.01), indicating that vaccination could stimulate the mice to produce an immune response. Among them, the IFN-γ levels in each dose group of the rBV-Cap-VP2 combined vaccine were higher than those in the rBV-Cap-VP2 single vaccine group and the rBV-VP2 single vaccine group, indicating that the combined vaccination of Cap and VP2 proteins could enhance the immune response. At the same time, the IFN-γ level in the 100 μg group of the rBV-Cap-VP2 combined vaccine was extremely significantly higher than those in the PCV2 commercial vaccine group and the SVA inactivated vaccine group (P < 0.01), indicating that the rBV-Cap-VP2 combined vaccine was superior in inducing IFN-γ.

[0060] The PCV2 and SVA IgG antibody levels in the sera of mice at 7, 14, 21, and 28 days after immunization were as Figure 7 shown: Specific antibodies against PCV2 could not be detected in the PBS control group. However, specific antibodies against PCV2 were successfully detected in the groups vaccinated with the rBV-Cap single vaccine, the 100 μg dose group of the rBV-Cap-VP2 combined vaccine, and the group vaccinated with the commercial PCV2 vaccine. This demonstrated that the vaccination we prepared could effectively stimulate the production of specific immune responses against PCV2 in mice. And with the increase in the immunization time, the antibody level reached its peak on the 21st day and showed a downward trend on the 28th day. The PCV2 specific antibody levels in the 100 μg group of the rBV-Cap-VP2 combined vaccine were higher than those in the rBV-Cap single vaccine group, indicating that the combined vaccination of Cap and VP2 proteins could enhance the immune response. Compared with the mice immunized with the commercial vaccine, the PCV2 specific antibody levels produced in the mice vaccinated with the 100 μg dose of the rBV-Cap-VP2 combined vaccine were significantly better (P < 0.01), indicating that the rBV-VP2-Cap combined vaccine might be superior to the commercial vaccine in inducing specific immune responses against PCV2.

[0061] After mice were inoculated with a single dose of rBV-VP2 vaccine or the BV-Cap-VP2 combined vaccine, they could effectively induce the production of high levels of specific IgG antibodies against the SVA VP2 protein. As the immunization time extended, the antibody level in the serum showed a trend of first increasing and then decreasing, and the highest antibody level was produced on the 21st day. Compared with the PBS control group, the antibody levels of the five groups of vaccinated mice showed extremely significant increases (P < 0.001). On the 21st day, the IgG antibody level produced by the 100 μg dose group of the rBV-Cap-VP2 combined vaccine was significantly higher than that of the SVA inactivated vaccine group. The antibody difference in the VP2 single vaccine group was extremely significantly higher than that of the 25 μg group of the rBV-Cap-VP2 combined vaccine, and in the combined vaccine, as the antigen amount increased, the antibody level was higher, indicating that the antibody level produced by the vaccine was related to the antigen amount. The level of the 50 μg group of the rBV-Cap-VP2 combined vaccine was slightly higher than that of the rBV-VP2 single vaccine group, indicating that the combined vaccine could better stimulate mice to produce specific antibodies. The SVA-specific antibody levels of the 50 μg and 100 μg groups of the rBV-Cap-VP2 combined vaccine were also higher than those of the SVA inactivated vaccine group, indicating that the rBV-Cap-VP2 combined vaccine was superior to the inactivated vaccine in inducing SVA-specific immune responses.

[0062] The PCV2 virus gene loads in various organs of mice were as Figure 8 shown. In mice inoculated with 100 μg dose of the rBV-Cap-VP2 combined vaccine, the copy numbers of the PCV2 virus genes detected in organs such as the liver, spleen, and lungs were at the lowest levels, and this data strongly indicated that the combined vaccine was the most superior in terms of immune protection. In these key visceral organs (such as the liver, spleen, and lungs), compared with mice inoculated with the commercial control vaccine, the PCV2 virus loads in the organs of mice inoculated with the combined vaccine were significantly reduced (P < 0.05). This confirmed that the novel rBV-Cap-VP2 combined vaccine had a more prominent protective effect on visceral organs and a statistically significant improvement compared with the existing commercial vaccines.

[0063] After SVA infection, no obvious clinical symptoms or deaths occurred in each group of mice. The sera of each group of mice were detected at 3, 6, 10, and 14 days after virus challenge, and the virus loads of the mice were detected by RT-qPCR. The results were as Figure 9As shown, with the passage of time, the virus in the serum of mice challenged with SVA first increased and then decreased, reaching a peak on the 6th day. Comparing each vaccine inoculation group with the PBS control group, the viral loads in the sera of all vaccine groups were significantly lower than those of the control group, which revealed that the vaccine had certain inhibitory activity against the SVA virus. Among the different dose groups of the combined vaccine, the 25 μg dose group showed a relatively high viral load, while the 50 μg dose group had a higher viral load than the 100 μg dose group. This phenomenon indicated that with the increase in antigen content, the immune effect was enhanced. Among all vaccine groups, the 100 μg group of the rBV-Cap-VP2 combined vaccine had the lowest viral load at 3, 6, 10, and 14 days. This result clearly demonstrated that the rBV-Cap-VP2 combined vaccine at this dose exhibited the best immune protection. Especially at 3 days and 14 days, the viral load of this group was extremely significantly different compared with the rBV-VP2 single vaccine and the SVA inactivated vaccine (P < 0.001).

[0064] The SVA virus loads in the organs of mice 6 days after challenge with SVA were as follows: the viral load in the lungs of mice was the highest, followed by the spleen and brain. This suggested that the SVA virus had strong replication ability in these organs. In the heart, spleen, kidney, intestine, and brain, the 100 μg of the rBV-Cap-VP2 combined vaccine was significantly lower than that of the rBV-VP2 single vaccine group (P < 0.05). This finding proved that the combined vaccine could more effectively inhibit the replication of the SVA virus in multiple organs compared with the single antigen vaccine. The viral copy number in the heart of the 100 μg group of the rBV-Cap-VP2 combined vaccine was significantly lower than that of the SVA inactivated vaccine, and slightly lower than that of the SVA inactivated vaccine group in other organs. Generally speaking, these data strongly supported that the rBV-Cap-VP2 combined vaccine had good systemic immune protection and might have a stronger protection advantage for specific organs.

[0065] There is currently no commercial SVA and PCV2 combined vaccine. Although Duan Weitong reported other experimental bivalent vaccines against SVA and PCV2 developed using different methods, there is only research on PCV2-VLP and SVA inactivated vaccines. In the present invention, the PCV2 Cap protein and the SVA VP2 protein were expressed by baculovirus, and recombinant single vaccines and combined vaccines of the two viruses were prepared. After immunizing mice for 28 days, the mice were challenged with SVA or PCV2, demonstrating that the combined vaccine has immunogenicity and protective effects in mice. The immune effect was evaluated using a mouse model. The results showed that both the single vaccine and the combined vaccine could produce PCV2 antibody levels similar to those of commercial vaccines; the rBV-Cap-VP2 combined vaccine had slightly better humoral and cellular immune results than the single vaccine group, probably because the combined vaccine would trigger immune synergy. When the two are used in combination, they can stimulate the immune system to produce a stronger and more persistent immune response than when either antigen is inoculated alone.

[0066] The above description is only for the embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. A PCV2-SVA bivalent vaccine, characterized in that, Using the PCV2 Cap protein and SVA VP2 protein with the nuclear localization signal removed as antigens; The amino acid sequence of the PCV2 Cap protein with the nuclear localization signal removed is shown in SEQ ID NO: 2; the amino acid sequence of the SVA VP2 protein is shown in SEQ ID NO: 3.

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