Porcine PRRSV and PCV2 recombinant adenovirus bivalent vaccine as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of vaccine preparation, and particularly relates to a porcine PRRSV (porcine reproductive and respiratory syndrome virus) and PCV2 (porcine circovirus 2) recombinant adenovirus bivalent vaccine as well as a preparation method and application thereof. The porcine PRRSV and PCV2 recombinant adenovirus bivalent vaccine provided by the invention comprises a recombinant adenovirus 1 for expressing PRRSV GP5, M and PCV2 TBCap proteins and a recombinant adenovirus 2 for expressing PRRSV GP2, GP3, GP4 and N proteins. After a piglet is immunized by the vaccine, high-level PRRSV and PCV2 specific antibodies and neutralizing antibodies can be induced to be generated, and the lymphocyte proliferation activity of the piglet and the expression level of cell factors IFN-gamma / IL-2 / IL-4 are also remarkably improved, so that the prepared vaccine can stimulate a body to generate humoral immune response and cellular immune response. The invention provides a direction for prevention and control of the PRRSV and the PCV2.

Description

Technical Field

[0001] This invention belongs to the field of vaccine preparation technology, specifically relating to a porcine PRRSV and PCV2 recombinant adenovirus bivalent vaccine, its preparation method, and its application. Background Technology

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly variable single-stranded RNA virus that causes severe respiratory symptoms in pigs and reproductive disorders in sows. The virus strains are highly variable, and its epidemic situation is complex. It can infect pigs of all sexes, ages, and breeds. A prominent feature of this disease is the reproductive dysfunction and respiratory symptoms it causes in pigs. Outbreaks often occur acutely, with extremely wide spread. The virus's rapid genetic evolution and significant antigenic variation make it difficult to eradicate effectively to date. Vaccination is currently the core strategy for prevention and control. Commercially available vaccines, including inactivated vaccines (KV), subunit vaccines, and live vector vaccines, all have shortcomings, failing to achieve an ideal balance between protective efficacy and safety. Therefore, the eradication of PRRS remains a significant challenge.

[0003] Porcine circovirus type 2 (PCV2) is a key pathogen causing porcine circovirus-related diseases such as multisystemic wasting syndrome in weaned piglets and reproductive disorders in sows. Its characteristic is inducing host immunosuppression, making secondary infections highly likely. Since its discovery, the main circulating strains of PCV2 have continuously evolved (from PCV2a to PCV2b, and more recently to PCV2d), with increasing genetic diversity. PCV2 often causes co-infection with other pathogens (such as Mycoplasma hyopneumoniae / MHP), significantly increasing the risk of infection and mortality. Vaccination is also an effective means of controlling PCV2. Commercially available vaccines include inactivated vaccines, subunit vaccines, and live vector vaccines, among which inactivated vaccines are widely used globally and are one of the important control tools.

[0004] Both PRRSV and PCV2 can cause reproductive disorders in sows and respiratory diseases in piglets. Clinically, PRRSV and PCV2 often occur as mixed infections, significantly reducing the survival rate and production performance of pigs by damaging both the respiratory and immune systems. Currently, the prevention and control of PRRSV and PCV2 still mainly rely on single vaccines, lacking a highly effective bivalent vaccine for both PRRSV and PCV2. Furthermore, existing single vaccines generally have the problem of limited viral type coverage and insufficient protective efficacy. In addition, the immunization schedules of different vaccines may interfere with each other, further affecting the overall immunization effect and resulting in unsatisfactory prevention and control outcomes. Summary of the Invention

[0005] In order to overcome the shortcomings and disadvantages of the prior art, the primary objective of this invention is to provide a bivalent vaccine of porcine PRRSV and PCV2 recombinant adenovirus.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned vaccine.

[0007] Another object of the present invention is to provide the application of the above-mentioned vaccine.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A bivalent vaccine of porcine PRRSV and PCV2 recombinant adenovirus, comprising recombinant adenovirus 1 expressing PRRSV GP5, M and PCV2 TBCap antigen proteins (GP5+M+TBCap) and recombinant adenovirus 2 expressing PRRSV GP2, GP3, GP4 and N antigen proteins (GP2+GP3+GP4+N);

[0010] The PRRSV GP5, M and PCV2 TBCap antigen proteins comprise P5+M(HP)+2A+GP5+M(CH-1a) fusion protein and GP5+M(NAD30)+2A+TBCap fusion protein, the amino acids of which are shown in SEQ ID NO:1 and 2, respectively.

[0011] The PRRSV GP2, GP3, GP4 and N antigen proteins include GP3+2A+GP4 fusion protein and PG2+2A+N fusion protein, whose amino acid sequences are shown in SEQ ID NO:3 and 4, respectively.

[0012] The nucleotide sequence encoding the GP5+M(HP)+2A+GP5+M(CH-1a) fusion protein is shown in SEQ ID NO:5; the nucleotide sequence encoding the GP5+M(NAD30)+2A+TBCap fusion protein is shown in SEQ ID NO:6.

[0013] The nucleotide sequence encoding the GP3+2A+GP4 fusion protein is shown in SEQ ID NO:7; the nucleotide sequence encoding the PG2+2A+N fusion protein is shown in SEQ ID NO:8.

[0014] In the vaccine, the volume ratio of recombinant adenovirus 1 to recombinant adenovirus 2 is 1:1;

[0015] The preferred dosage concentration of recombinant adenovirus 1 and recombinant adenovirus 2 in the vaccine is 1.0 × 10⁻⁶. 8 TCID 50 / mL;

[0016] The method for preparing the porcine PRRSV and PCV2 recombinant adenovirus bivalent vaccine includes the following steps:

[0017] (1) Insert the nucleotide sequence encoding the GP5+M(HP)+2A+GP5+M(CH-1a) fusion protein and the nucleotide sequence encoding the GP5+M(NAD30)+2A+TBCap fusion protein into an adenovirus shuttle plasmid containing dual promoters, wherein the P5+M(HP)+2A+GP5+M(CH-1a) fusion protein and the GP5+M(NAD30)+2A+TBCap fusion protein are each expressed using one promoter, thus obtaining adenovirus shuttle plasmid 1;

[0018] (2) Insert the nucleotide sequence encoding the GP3+2A+GP4 fusion protein and the nucleotide sequence encoding the PG2+2A+N fusion protein into an adenovirus shuttle plasmid containing dual promoters, wherein the GP3+2A+GP4 fusion protein and the PG2+2A+N fusion protein are each expressed using one promoter, to obtain adenovirus shuttle plasmid 2.

[0019] (3) After linearizing adenovirus shuttle plasmid 1 and adenovirus shuttle plasmid 2, they were co-transformed with adenovirus backbone vector into homologous recombination competent cells suitable for adenovirus vector construction to obtain recombinant adenovirus plasmids.

[0020] (4) The recombinant adenovirus plasmid was packaged to obtain recombinant adenovirus 1 expressing PRRSV GP5, M and PCV2 TBCap antigen proteins and recombinant adenovirus 2 expressing PRRSV GP2, GP3, GP4 and N antigen proteins, respectively.

[0021] (5) Mix the recombinant adenovirus 1 expressing PRRSV GP5, M and PCV2 TBCap antigen proteins obtained in step (4) with the recombinant adenovirus 2 expressing PRRSV GP2, GP3, GP4 and N antigen proteins to obtain a porcine PRRSV and PCV2 recombinant adenovirus bivalent vaccine.

[0022] The adenovirus shuttle plasmid mentioned in step (1) is preferably pAdTrack;

[0023] The adenovirus backbone vector mentioned in step (3) is preferably pAdEasy-1;

[0024] The linearization process described in step (3) uses PacⅠ enzyme;

[0025] The preferred receptive state in step (3) is the BJ-5183 receptive state;

[0026] The host cells used for packaging in step (4) are preferably HEK 293 cells;

[0027] The preferred specific packaging operation in step (4) is as follows:

[0028] The recombinant adenovirus plasmid was co-transfected with the transfection reagent into the cells. After 7-10 days of transfection, the culture medium and cells were collected. The collected culture medium and cells were repeatedly frozen and thawed, centrifuged, and the supernatant was collected to obtain the recombinant adenovirus.

[0029] The transfection reagent is Lipofectamine. TM 3000 transfection reagents;

[0030] The application of the porcine PRRSV and PCV2 recombinant adenovirus bivalent vaccine in the preparation of products for the prevention and treatment of porcine PRRSV or PCV2;

[0031] The present invention has the following advantages and effects compared with the prior art:

[0032] (1) This invention selects the GP5 protein, which can induce PRRSV neutralizing antibodies, the M protein, which participates in viral assembly, and the GP2, GP3, GP4, and N proteins, which have T and B cell epitopes, as candidate antigen proteins for constructing PRRSV vaccines; and selects the Cap protein and adds T / B cell antigen epitopes as the target antigen protein for preparing PCV2 vaccines. Then, a multi-epitope gene cassette is constructed by tandem linking the foot-and-mouth disease virus 2A peptide, and four recombinant adenoviruses are successfully constructed using the AdEasy adenovirus vector system: rAd-TBCap, rAd-GP5+M, rAd-GP5+M+TBCap, and rAd-GP3+GP4+GP2+N. Western blotting detection of the target protein expression showed that the constructed recombinant adenoviruses could express the target protein of the expected size after infecting HEK293 cells; indirect immunofluorescence further confirmed the successful expression of the target protein in the cells. After continuous passage of the recombinant adenovirus to the F10 generation, the viral passage stability test showed that the gene sequence of the target protein did not change, indicating that the recombinant adenovirus has good stability.

[0033] (2) The porcine PRRSV and PCV2 recombinant adenovirus bivalent vaccine provided by the present invention contains recombinant adenovirus rAd-GP5+M+TBCap and rAd-GP2+GP3+GP4+N. After immunizing piglets with this vaccine, high levels of PRRSV and PCV2 specific antibodies and neutralizing antibodies can be induced. Furthermore, the proliferation activity of piglet lymphocytes and the expression levels of cytokines IFN-γ / IL-2 / IL-4 are also significantly increased, suggesting that the prepared vaccines can stimulate the body to produce humoral immune responses and cellular immune responses.

[0034] (3) The present invention prepared a bivalent vaccine of recombinant adenovirus PRRSV and PCV2 with good immunization effect, which provides a theoretical reference for the prevention and control of PRRSV and PCV2. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the recombinant adenovirus sequence design in this invention.

[0036] Figure 2 This is a map of the recombinant adenovirus shuttle plasmid, where a: pAdTrack-CMV-MCS (empty vector), b: pAdTrack-TBCap, c: pAdTrack-GP5+M, d: pAdTrack-GP5+M+TBCap, and e: pAdTrack-GP3+GP4+GP2+N.

[0037] Figure 3 This is a diagram showing the PCR identification results of the recombinant adenovirus shuttle plasmid.

[0038] Figure 4 This is a graph showing the results of PCR linearization identification of recombinant adenovirus shuttle plasmids. Among them, 1, 3, 5 and 7: pAdTrack-TBCap, pAdTrack-GP5+M, pAdTrack-GP5+M+TBCap and pAdTrack+GP2+GP3+GP4+N that were not successfully digested with enzymes; 2, 4, 6 and 8: pAdTrack-TBCap, pAdTrack-GP5+M, pAdTrack-GP5+M+TBCap and pAdTrack+GP2+GP3+GP4+N that were successfully digested with enzymes; M: DL2000 DNA marker.

[0039] Figure 5 This is a diagram showing the results of PacI restriction enzyme digestion identification of the recombinant adenovirus plasmid. For a, 1: pAdEasy-TBCap, 2: AdEasy-GP5+M; for b, 1: pAdEasy-GP5+M+TBCap; 2: pAdEasy-GP2+GP3+GP4+N; M: DL15000 DNA marker.

[0040] Figure 6 This is a display of HEK293 cells after transfection with recombinant adenovirus plasmid. In the image, a: pAdEasy-TBCap, b: pAdEasy-GP5+M, c: pAdEasy-GP5+M+TBCap, d: pAdEasy-GP2+GP3+GP4+N; M: blank control (HEK293).

[0041] Figure 7This is a graph showing the PCR identification results of the recombinant adenovirus genome. In the graph, 1: rAd-TBCap, 2-3: rAd-GP5+M; 4-5: rAd-GP5+M+TBCap, 6-7: rAd-GP2+GP3+GP4+N, and M: DL 5000 DNA marker.

[0042] Figure 8 This is a graph showing the results of Western blot analysis of the recombinant protein.

[0043] Figure 9 This is a graph showing the results of indirect immunofluorescence detection of exogenous gene expression. In the graph, a: mouse His-tagged antibody, b: mouse HA-tagged antibody, and c: mouse FLAG-tagged antibody.

[0044] Figure 10 This is a graph showing the stability test results of recombinant adenovirus at passages F5 and F10. In the graph, a represents passage F5, b represents passage F10, 1 represents rAd-TBCap, 2-3 represent rAd-GP5+M, 4-5 represent rAd-GP5+M+TBCap, 6-7 represent rAd-GP3+GP4+GP2+N, and M represents DL5000 DNA marker.

[0045] Figure 11 This is a schematic diagram of the immunization and challenge program for piglets.

[0046] Figure 12 This is a graph showing the results of detection of PRRSV and PCV2 specific antibodies in piglet serum.

[0047] Figure 13 This is a graph showing the results of the detection of neutralizing antibody titers against PRRSV and PCV2 in peripheral blood of piglets.

[0048] Figure 14 This is a graph showing the detection results of cytokines in piglet serum. a: IFN-γ, b: IL-2, c: IL-4, 1: rAd-GP5+M+TBCap; 2: rAd-GP2+GP3+GP4+N+rAd-GP5+M+TBCap; 3: Commercial vaccine; 4: DMEM; One-way ANOVA: ns, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0049] Figure 15This is a graph showing the results of peripheral blood lymphocyte proliferation activity detection in piglets. a: PRRSV, b: PCV2, 1: rAd-GP5+M+TBCap, 2: rAd-GP2+GP3+GP4+N+rAd-GP5+M+TBCap, 3: commercial vaccine, 4: DMEM. One-way ANOVA: ns, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0050] Figure 16 These are images showing some of the clinical symptoms in piglets.

[0051] Figure 17 This is a chart analyzing the survival rate results of virus attack protection. Detailed Implementation

[0052] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0053] In this embodiment, GraphPad Prism 9.5.1 software was used to conduct statistical analysis on the inter-group data, specifically employing one-way ANOVA and two-way ANOVA methods. During the analysis, a result was considered statistically significant if the obtained probability (P) value was less than 0.05.

[0054] Example 1

[0055] I. Experimental Methods

[0056] 1. Design of target gene sequence

[0057] (1) The Cap gene in the ORF2 region was found based on the PCV2LG strain in GenBank (accession number: MW303946.1). The base sequence of T and B cell antigen epitopes and 6×His tag protein was introduced into the Cap gene, and finally the recombinant PCV2-TBCap gene sequence was obtained. Based on the PRRSV sequences in GenBank, the GP5 and M gene sequences of HP-PRRSV (accession number: EF112445.1), NADC30-Like (accession number: JN654459.1), and CH-1a (accession number: AY032626.1) were identified and linked with a flexible linker peptide and P2A. The sequences were codon-optimized, and a base sequence encoding a HA tag protein was added to the end of GP5+M (HP), a base sequence encoding a 3×FLAG tag protein was added to the end of GP5+M (CH-1a), and a base sequence encoding a 6×His tag protein was added to the end of GP5+M (NAD30), respectively, to obtain the recombinant GP5+M gene sequences of HP-PRRSV, CH-1a, and NAD30-Like. Based on the PRRSV sequences published in GenBank, the GP2, N, GP3, and GP4 gene sequences of NADC30-Like (accession number: JN654459.1) were found. P2A was used to link the GP2 and N genes and the GP3 and GP4 genes, respectively. The sequences were codon optimized and the base sequence encoding the 6×His tag protein was added to the end to obtain the NAD30-Like recombinant GP2+2A+N and GP3+2A+GP4 gene sequences.

[0058] (2) The above-mentioned target gene sequence and adenovirus shuttle vector pAdTrack-CMV (commercially available) were synthesized by Suzhou Hongxun Biotechnology Co., Ltd., wherein:

[0059] PCV2-TBCap is ligated to pAdTrack-CMV via restriction enzyme sites KpnⅠ and HindⅢ to obtain pAdTrack-TBCap;

[0060] GP5+M(HP)+2A+GP5+M(CH-1a) is linked to pAdTrack-CMV via restriction sites KpnⅠ and HindⅢ, and GP5+M(NAD30) is linked to pAdTrack-GP5+M via restriction sites ClaⅠ and SpeⅠ.

[0061] GP5+M(HP)+2A+GP5+M(CH-1a) is linked to pAdTrack-CMV via restriction sites KpnⅠ and HindⅢ, and GP5+M(NAD30)+2A+TBCap is linked to pAdTrack-CMV via restriction sites ClaⅠ and SpeⅠ, resulting in pAdTrack-GP5+M+TBCap.

[0062] GP3+2A+GP4 was ligated to pAdTrack-CMV via restriction sites KpnⅠ and HindⅢ, and PG2+2A+N was ligated to pAdTrack-CMV via restriction sites ClaⅠ and SpeⅠ, to obtain pAdTrack-GP3+GP4+GP2+N( Figure 1 and Figure 2 ).

[0063] Nucleotide sequence of PCV2-TBCap:

[0064] ATGACATACCCACGCAGAAGATACAGACGCAGAAGACACAGACCCAGATCACATCTGGGACAGATCCTGAGAAGAAGACCTTGGCTGGTGCATCCAAGACTGAGATATCGCTGGAGACGCAAGAACGGAATCTTCAACACCCGCCTGTCAAGAACCATCGGATACACAGTGAAGGCCACAACAGTGAGAACACCATCTTGGGCAGTGGACATGATGCGCTTCAACATCAACGACTTCCTGCCACCAGGAGGAGGATCAAATCCACTGACAGTGCCATTCGAGTACTACAGAATCCGCAAGGTGAAGGTGGAGTTTTGGCCTTGTTCACCCATCACACAGGGAGATAGAGGAGTCGGATCAACAGCAGTGATCCTGGACGACAACTTCGTGACAAAGGCAAACGCACTGACATACGACCCATACGTGAACTACTCCTCCAGACACACCATCCCACAGCCCTTCTCATACCACTCAAGATACTTCACACCCAAACCCGTGCTGGATAGAACAATCGACTACTTCCAGCCCAACAACAAGAGAAACCAGCTCTGGCTGAGACTGCAGACAACAGGAAACGTGGATCACGTGGGACTGGGAACAGCATTCGAGAACTCCAAGTACGACCAGGACTACAACATCCGCATCACCATGTACGTGCAGTTCAGAGAGTTCAACCTGAAAGACCCCCCACTGAATCCAAAACACCACCATCACCACCACTAA

[0065] Amino acid sequence of PCV2-TBCap:

[0066] MTYPRRRYRRRRHRPRSHLGQILRRRPWLVHPRLRYRWRRKNGIFNTRLSRTIGYTVKATTVRTPSWAVDMMRFNINDFLPPGGGSNPLTVPFEYYRIRKVKVEFWPCSPITQGDRGVGS TAVILDDNFVTKANALTYDPYVNYSSRHTIPQPFSYHSRYFTPKPVLDRTIDYFQPNNKRNQLWLRLQTTGNVDHVGLGTAFENSKYDQDYNIRITMYVQFREFNLKDPPLNPKHHHHHH*

[0067] GP5+M(HP)+2A+GP5+M(CH-1a) nucleotide sequence:

[0068]

[0069] P5+M(HP)+2A+GP5+M(CH-1a) amino acid sequence:

[0070]

[0071] GP5+M (NAD30) nucleotide sequence (SEQ ID NO:3):

[0072]

[0073] GP5+M (NAD30) amino acid sequence:

[0074] MLGKCSTAGCCSQLPFLWCIVPFCFAALVNANSNSSSNLQLIYNLTICELNGTDWLDKKFSWAVETFVIFPVLTHIVSYGALTTSHFLDTVGLVTVSAAGYFHERYVLSSIYAVCALAALVCFAIRLAKNCMSWRYSCTRYTNFLLDTKGKLYRWRSSVIIEKGGKVDVEGHLIDLKRVVLDGSAATPVTKI SAEQWGRPGPGPGGSSIDDFCNDSTAVQKVLLAFSITYTPIMIYALKVSRGRLLGLLHLLIFLNCAFTFGYMTFVHFQSTNRVALTMGAVVALLWGVYSAMETWRFIASRCRLCLLGRKYILAPAHHVESAAGFHPITANDNHAFVVRRPGSTTVNGTLVPGLKSLVLGGRRAVKRGVVNLVKYAKHHHHHH*

[0075] The nucleotide sequence of GP5+M(NAD30)+2A+TBCap:

[0076]

[0077] The amino acid sequence of GP5+M(NAD30)+2A+TBCap:

[0078]

[0079] The nucleotide sequence of GP3+2A+GP4:

[0080]

[0081]

[0082] The amino acid sequence of GP3+2A+GP4:

[0083]

[0084] The nucleotide sequence of PG2+2A+N:

[0085]

[0086] The amino acid sequence of PG2+2A+N:

[0087]

[0088]

[0089] Note: The horizontal lines represent flexible linker peptides, the wavy lines represent P2A, the double underlines represent HA-tagged proteins, and the dashed underlines represent FLAG-tagged proteins.

[0090] 2. Obtaining recombinant adenovirus shuttle plasmid

[0091] (1) Identification of recombinant adenovirus shuttle plasmid: Using the synthesized recombinant shuttle plasmid as a template, Primer 5.0 software was used to identify the recombinant adenovirus shuttle plasmid according to... Figure 1 Primers were designed for the gene sequences (Table 1), and the target gene sequences were identified by PCR.

[0092] Table 1 Primer sequences

[0093]

[0094] (2) Amplification and extraction of recombinant adenovirus shuttle plasmid

[0095] The successfully identified recombinant shuttle plasmids pAdTrack-TBCap, pAdTrack-GP5+M, pAdTrack-GP5+M+TBCap, and pAdTrack-GP3+GP4+GP2+N were transformed into DH5α competent cells, as detailed in the instruction manual. White single colonies were picked from agar plates and inoculated into 5 mL of LB liquid medium (Amp). After incubation at 37°C and 200 rpm for 6 h, non-endotoxin-free mini-preparations were performed. The extracted plasmids were amplified by PCR, and the amplification products were identified by electrophoresis. The selected positive clones were preserved and sequenced, and named pAdTrack-TBCap, pAdTrack-GP5+M, pAdTrack-GP5+M+TBCap, and pAdTrack-GP3+GP4+GP2+N, respectively.

[0096] 3. Linearization of recombinant adenovirus shuttle plasmid

[0097] The constructed shuttle plasmid was linearized by digestion with Thermo Fisher Scientific's PmeI rapid digestion enzyme at 37°C for 6 hours. The digestion system consisted of: 2 μL PmeI, 5 μL 10× Buffer, 15 μg target plasmid, and ddH2O to a final volume of 50 μL. Based on the PmeI restriction sites, a fragment of approximately 453 bp was selected, and primers designed using Primer 5 were used for linearization identification. The primers are shown in Table 1.

[0098] 4. Construction of recombinant adenovirus plasmid

[0099] The successfully identified linearized shuttle plasmids pAdTrack-TBCap, pAdTrack-GP5+M, pAdTrack-GP5+M+TBCap, and pAdTrack-GP3+GP4+GP2+N were co-transformed into BJ-5183 competent cells (Shanghai Weidi Biotechnology) with the adenovirus backbone vector pAdEasy-1 (Takara Bio Engineering (Dalian) Co., Ltd.), respectively, following the manufacturer's instructions. The recombinant bacterial culture was initially verified by Pac-I digestion. The digestion system consisted of: 1 μL Pac-I, 2.5 μL 10×Buffer, 7.5 μg target plasmid, and ddH2O to a final volume of 25 μL. The bacterial cultures corresponding to the recombinant plasmids identified by Pac-I were transferred to 250 mL Erlenmeyer flasks and cultured at 37°C and 200 rpm for 24–36 h on a shaker. After culture, plasmid extraction was performed according to the plasmid extraction kit (omega); the concentrations of pAdEasy-TBCap, pAdEasy-GP5+M, pAdEasy-GP5+M+TBCap, and pAdEasy-GP2+GP3+GP4+N recombinant adenovirus plasmids were measured.

[0100] 5. Linearization of recombinant adenovirus plasmids

[0101] For the recombinant adenovirus plasmid, PacI single-enzyme digestion was used to linearize it. The specific digestion system is as described in step 4, with three replicates for each digestion system. The constructed digestion reaction system was incubated overnight in a 37°C water bath. After the reaction, 0.8% agarose gel electrophoresis was performed to observe the band size, followed by chloroform extraction to recover and purify the DNA.

[0102] 6. Obtaining recombinant adenovirus

[0103] (1) Using Lipofectamine from Thermo Fisher Scientific TM 3000 transfection reagents were packaged into HEK293 cells (Guangzhou Lide Biotechnology Co., Ltd.). Specifically, the recombinant adenovirus plasmids pAdEasy-TBCap, pAdEasy-GP5+M, pAdEasy-GP5+M+TBCap, and pAdEasy-GP2+GP3+GP4+N obtained in step 5 were transfected into HEK 293 cells, respectively. The transfection procedure followed Thermo Fisher Scientific's transfection instructions. Note that during virus packaging, approximately 1 / 5 of the complete culture medium should be added every three days to replenish lost medium and prevent cell drying. 7–10 days post-transfection, when obvious cytopathic effect (CPE) or fluorescence is observed, the culture medium and cells are collected.

[0104] (2) Repeat the freeze-thaw cycle of the culture medium and cells collected in step (1) at -80℃ / 37℃ 3 times. Take the freeze-thaw solution into a sterile centrifuge tube and centrifuge at 4℃ and 11000rpm for 10min. The collected supernatant is the obtained recombinant adenovirus rAd-TBCap, rAd-GP5+M, rAd-GP5+M+TBCap, and rAd-GP2+GP3+GP4+N.

[0105] 7. Identification of recombinant adenovirus genome by PCR

[0106] Viral DNA and RNA were extracted using the Viral DNA Extraction Kit (D3892-02) and the Viral RNA Extraction Kit (R6872-02), respectively. The RNA was reverse-transcribed using Takara reverse transcriptase to obtain mRNA. Using DNA and mRNA as templates, the target gene was detected according to the primers in Table 1.

[0107] 8. Passaging of recombinant adenovirus

[0108] The titer of the first-generation recombinant adenovirus is relatively low. To increase the titer, it is necessary to gradually increase the amount of virus-coated cells. The procedure starts with using a six-well plate, transitions to a 60mm cell culture dish, and then to a 100mm cell culture dish. In the infection process, firstly, prepare HEK 293 cells in good condition. When the cells grow to about 70-80% confluence, starve them for 2 hours. Then, take 300μL of the first-generation virus solution (the supernatant collected in step 6(2)) to infect HEK 293 cells and place them in an environment of 37℃ and 5% CO2 for 6 hours. After 6 hours, discard the virus solution, wash the cells with PBS 1-2 times, and then change to maintenance medium for culture. Closely record the cytopathic effect (CPE) status. After 7-10 days, when the degree of cytopathic effect reaches 70-80%, obtain the second-generation virus according to step 6(2). Subsequent passages are performed in the same manner to obtain a high-titer recombinant adenovirus.

[0109] 9. Determination of recombinant adenovirus titer

[0110] The third-generation recombinant adenovirus viral solution obtained from packaging was serially diluted 10-fold to obtain different dilutions from 10⁻¹ to 10⁻¹⁰, and inoculated into HEK 293 cells. Eight replicates were performed for each dilution, and negative control wells were also included. 100 μL of the corresponding dilution was added to each well. CPE (cytotoxicity in vitro) was closely observed in each well for 96 hours after inoculation. Finally, the median tissue culture infectious dose (TCID) of the recombinant adenovirus was calculated using the Reed-Muench method. 50 ).

[0111] II. Test Results

[0112] 1. Identification of recombinant adenovirus shuttle plasmid

[0113] Figure 3 This image shows the PCR identification results of recombinant adenovirus shuttle plasmids pAdTrack-TBCap, pAdTrack-GP5+M, pAdTrack-GP5+M+TBCap, and pAdTrack+GP2+GP3+GP4+N. All of these recombinant adenovirus shuttle plasmids showed specific bands at the expected locations, indicating successful construction of the recombinant adenovirus shuttle plasmids. Figure 3 ).

[0114] 2. Construction of recombinant adenovirus plasmid

[0115] (1) Linearization identification of recombinant adenovirus shuttle plasmid

[0116] A pair of specific primers were designed around the PmeI restriction site for amplification and identification, with an amplified band size of approximately 453 bp. If a band of the corresponding size was amplified, linearization was unsuccessful; if no band of the corresponding size was amplified, linearization was successful due to PmeI cleavage. 1% agarose gel electrophoresis results showed (…). Figure 4 The uncut bands amplified to approximately 453 bp in size (lanes 1, 3, 5, and 7), while the successfully linearized bands were bandless (lanes 2, 4, 6, and 8). These results are consistent with expectations, indicating that the recombinant shuttle adenovirus plasmid has been successfully linearized.

[0117] (2) Identification of recombinant adenovirus plasmid

[0118] After linearizing the recombinant adenovirus shuttle plasmid, it was introduced into BJ5183 competent cells containing the adenovirus backbone plasmid pAdEasy-1. Following homologous recombination, recombinant adenovirus plasmids were successfully obtained: pAdEasy-TBCap, pAdEasy-GP5+M, pAdEasy-GP5+M+TBCap, and pAdEasy-GP2+GP3+GP4+N. Positive recombinant adenoviruses were identified by PacI enzyme digestion. As expected, two bands should appear after digestion, one greater than 15000 bp and the other 3000 bp or 4500 bp. Detection was performed using 0.8% agarose gel electrophoresis. The results showed that... Figure 5 The band size obtained was consistent with the expected result, which fully confirms the successful construction of the recombinant adenovirus plasmid.

[0119] 3. Obtaining recombinant adenovirus

[0120] (1) Pathological changes in transfected cells

[0121] Linearized recombinant adenovirus plasmids pAdEasy-TBCap, pAdEasy-GP5+M, pAdEasy-GP5+M+TBCap, and pAdEasy-GP2+GP3+GP4+N were transfected into normal HEK 293 cells. Cell status was continuously observed for 7-11 days post-transfection. Results showed that over time, cells gradually exhibited a shrunken, rounded morphology and began to aggregate. In more severe cases, cells even ruptured and detached from the bottom of the culture dish. Figure 6 In stark contrast, normal cells not inoculated with the recombinant adenovirus plasmid did not exhibit any of the aforementioned abnormal changes. Based on this, the virus was successfully harvested (P1 generation) by repeatedly freezing and thawing the cells that showed significant changes. Subsequently, the harvested virus was used again to infect HEK 293 cells, and they were continuously passaged.

[0122] (2) Recombinant adenovirus genome PCR identification

[0123] Using the extracted recombinant adenovirus genome sequence as a template, PCR amplification was performed using primers listed in Table 1: TBCap-F / R, HC5M-F / R, NAD30-F / R, NACap-F / R, GP34-F / R, and GP2N-F / R. The results of 1% agarose gel electrophoresis confirmed that... Figure 7 The presence of specific bands at approximately 700bp, 2452bp, 1170bp, 2452bp, 1962bp, 1531bp, and 1263bp, consistent with the expected band sizes, indicates that the recombinant adenovirus was successfully constructed.

[0124] 4. Titer determination of recombinant adenovirus

[0125] The titer of recombinant adenovirus was calculated using the Reed-Muench method, and the titer of the 5th generation rAd-TBCap was 10. 9.67 TCID 50 / mL, rAd-GP5-M titer is 10 9.64 TCID 50 / mL, rAd-GP5-M-CAP titer is 10 10.33 TCID 50 / mL, rAd-GP3-GP4-GP2-N titer is 10 10.82 TCID 50 / mL.

[0126] Example 2

[0127] I. Experimental Methods

[0128] 1. Expression and identification of recombinant proteins

[0129] (1) Western Blot: 200 μL of cell samples of the third generation recombinant adenovirus rAd-TBCap, rAd-GP5+M, rAd-GP5+M+TBCap and rAd-GP2+GP3+GP4+N were collected into 1.5 mL EP tubes, 50 μL of 5× protein loading buffer (Shanghai Yamei Biomedical Technology Co., Ltd.) were added, and the mixture was heated in a metal bath at 100℃ for 10 min to prepare protein samples. Protein samples were added to a 10% (v / v) SDS-PAGE gel for electrophoresis. Appropriately sized PVDF membranes were then attached to the corresponding protein sites and electrophoretically transferred to the membrane at 200 mA for 90 min. The PVDF membranes were blocked with 5% skim milk powder and washed after blocking. Primary antibodies against mouse HA, FLAG, and His (1:1000 dilution) and secondary antibodies against goat anti-mouse IgG-HRP were used for incubation. The PVDF membranes were then developed using ECL chemiluminescence solution. Mouse His and Flag monoclonal antibodies were purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., mouse HA monoclonal antibody from Abcam (UK), and goat anti-mouse IgG-HRP antibody from Shanghai Beyotime Biotechnology Co., Ltd.

[0130] (2) Indirect immunofluorescence: HEK293 cells in logarithmic growth phase and in an adherent growth state were infected with third-generation recombinant adenoviruses pAd-TBCap, pAd-GP5+M, pAd-GP5+M+TBCap, and pAd-GP2+GP3+GP4+N at an MOI of 1. Empty cells supplemented with DMEM were used as a control group. The cells were cultured at 37°C. When 80% CPE was observed in the infected HEK293 cells, primary antibodies (mouse-derived His-tagged antibody, HA-tagged antibody, and FLAG-tagged antibody) were added and incubated overnight at 4°C. The cells were washed with PBS the next day, and then incubated with secondary antibody (FITC-labeled goat anti-mouse IgG antibody diluted 1:1000) for 1 hour. The cells were then observed and photographed under a fluorescence microscope. IgG-FITC was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0131] 2. Stability assay of recombinant adenovirus after passage

[0132] Once the HEK 293 cells reached 80% confluence, following Example 1, the virus fluid harvested initially (P1 generation virus fluid) was passaged repeatedly. 250 μL of the virus fluid from the 5th and 10th generations were taken from each cell to extract viral RNA and DNA for PCR identification.

[0133] II. Test Results

[0134] (1) Identification of recombinant adenovirus protein

[0135] HEK 293 cell samples infected with the obtained third-generation recombinant adenoviruses rAd-TBCap, rAd-GP5+M, rAd-GP5+M+TBCap, and rAd-GP3+GP4+GP2+N were first subjected to SDS-PAGE gel electrophoresis, then transferred to PVDF membranes, and identified by Western blot to detect the expression of the target proteins. In the identification process, mouse HA, FLAG, and His-tagged antibodies were used as primary antibodies, and FITC-labeled goat anti-mouse IgG-HRP antibody was used as the secondary antibody. Results are as follows: Figure 8 As shown: Figure 8 The protein sizes in lane 1 of a, b, and c are 43 kDa, which corresponds to the protein sizes of HP-PRRSV, NADC30-Like, and CH-1a strains in rAd-GP5+M, respectively, proving successful expression. Figure 8 The protein sizes in lane 2 of a, b, and c are 43 kDa, which corresponds to the GP5+M protein sizes of HP-PRRSV, NADC30-Like, and CH-1a strains in rAd-GP5+M+TBCap, respectively, proving successful expression. Figure 8 The size of the protein in lane c is consistent with the size of the TBCap protein in rAd-TBCap, with a distinct specific band appearing at approximately 43 kDa; Figure 8 Lanes d, e, and f (lane 1) show that cell samples infected with rAd-GP3+GP4+GP2+N exhibited specific bands at approximately 15 kDa, 21 kDa, 34 kDa, and 37 kDa, respectively, consistent with the protein sizes of GP2, GP3, GP4, and N. The remaining lanes contained blank cells with no specific bands. These results indicate that the recombinant adenovirus constructed in this invention can express the target protein of the expected size after infection of HEK 293 cells.

[0136] Indirect immunofluorescence was used to detect the infection status of recombinant adenovirus and the expression of the recombinant target protein. The experimental results showed... Figure 9 HEK 293 cells infected with the recombinant adenovirus exhibited specific green fluorescence, while no fluorescence was observed in the negative control wells. This result demonstrates that the recombinant adenovirus was successfully constructed and can effectively infect HEK 293 cells.

[0137] (2) Stability assay of recombinant adenovirus passage

[0138] The genomic sequences of recombinant adenoviruses from generations 5 and 10 were selected as templates. Primers TBCap-F / R, HC5M-F / R, NAD30-F / R, NACap-F / R, GP34-F / R, and GP2N-F / R were used for PCR amplification. After amplification, the amplified products were detected by 1% agarose gel electrophoresis. The results showed specific bands at approximately 700 bp, 2452 bp, 1170 bp, 1962 bp, 1531 bp, and 1263 bp. Figure 10 The size of these bands matched the expected size of the target gene. Sequencing of these bands showed that the target gene remained stably expressed after passage of the recombinant adenovirus.

[0139] Example 3

[0140] I. Experimental Methods

[0141] 1. Immunization evaluation test for piglets

[0142] Twenty 4-week-old piglets (5 piglets / group) with negative serological tests for PRRSV and PCV were selected from large-scale pig farms in Guangdong Province. Immunization grouping and dosage are shown in Table 2. Immunization was performed via intramuscular injection, with a booster immunization given 14 days after the initial immunization. Peripheral blood was collected from piglets at 0, 14, 28, and 35 days, and serum was separated for the determination of specific antibodies, neutralizing antibodies, cytokines, and lymphocyte proliferation activity. The immunization and challenge procedures for piglets are detailed below. Figure 11 (Drawn by Figdraw) The animal experimental procedures were approved by the Experimental Animal Ethics Committee of South China Agricultural University (2024f289). The commercially available PRRSV vaccine was purchased from Wuhan Keqian Biotechnology Co., Ltd., and the commercially available PCV2 vaccine was purchased from China Animal Husbandry Industry Co., Ltd. All piglets were PRRSV and PCV2 antibody-negative before the first immunization. The 10th generation recombinant adenoviruses pAd-GP5+M+TBCap and pAd-GP2+GP3+GP4+N obtained in Example 1 were mixed at a volume ratio of 1:1, with a concentration of 1.0 × 10⁻⁶ after mixing. 8 TCID 50 / mL, as group 2.

[0143] Table 2 Grouping of Animal Experiments

[0144]

[0145] 2. Detection of PRRSV and PCV2-specific antibodies in piglet serum

[0146] The levels of PRRSV / PCV2 specific antibodies in the peripheral blood of piglets were determined using the porcine reproductive and respiratory syndrome (PRRS) antibody ELISA kit (RXYB0002) and porcine circovirus 2-dCap-ELISA kit (RX-AN500680P) manufactured by Shenzhen Fende Biotechnology Co., Ltd. For detailed operating procedures, please refer to the instruction manual.

[0147] 3. Detection of neutralizing antibodies against PRRSV and PCV2 in peripheral serum of piglets

[0148] Serum samples were inactivated at 56°C for 30 min, then diluted twofold. 50 μL of the serum diluent was mixed with a solution containing 100 TCID50. 50 Mix 0.1 mL of PRRSV or PCV2 (NADC30-like PRRSV strain and PCV2 d strain, provided by the Veterinary Microbiology and Immunology Laboratory of South China Agricultural University) with an equal volume of viral supernatant and incubate at 37°C for 1 h. Wash Marc-145 or PK-15 cells (provided by the Veterinary Microbiology and Immunology Laboratory of South China Agricultural University) twice with PBS in 96-well plates, cover the serum-virus mixture in eight aliquots, and culture in maintenance medium supplemented with trypsin. After incubation at 37°C for 1 h, replace the mixture with 100 μL of fresh maintenance medium. Perform indirect immunofluorescence on PK-15 cells. PCV2 and PRRSV monoclonal antibodies were purchased from Guangzhou Qianxun Biotechnology Co., Ltd. Marc-145 cells were observed directly with the naked eye and under a microscope for cytopathic effect (CPE). The highest serum dilution at which 50% cytopathic effect was observed was the neutralizing antibody titer.

[0149] 4. Detection of peripheral blood cytokines in piglets

[0150] The secretion levels of IFN-γ, IL-2 and IL-4 in piglets 28 days after their first immunization were measured using the Jiangsu Enzyme Immunized Pig IFN-γ ELISA Kit (MM-041202), Pig IL-2 ELISA Kit (MM-042102) and Pig IL-4 ELISA Kit (MM-041902). The ELISA operation procedures are as described in the instruction manual.

[0151] 5. Detection of peripheral blood lymphocyte proliferation activity in piglets

[0152] To investigate the effect of vaccine immunization on the proliferative activity of porcine lymphocytes, lymphocytes were isolated from peripheral blood of piglets on day 35 post-primary immunization according to the instructions of the Haoyang TBD porcine peripheral blood lymphocyte isolation kit. Lymphocyte proliferative activity was measured using the CCK-8 assay. Piglet peripheral blood lymphocytes were divided into a positive control group, a negative control group, and an experimental group, with four replicates per group. Specific operating procedures were detailed in the instruction manual. The cells were incubated at 37℃ in a 5% CO2 incubator for 2–4 hours, with OD measured every 30 minutes.450 Value. The Stimulation Index (SI) is calculated using the following formula:

[0153]

[0154] 6. PRRSV challenge protection test

[0155] Two weeks after the completion of the second immunization, select piglets and inject 1 mL of virus with a titer of 10 into the neck muscle. 6 TCID 50 / mL of NADC30-like PRRSV strain (provided by the Veterinary Microbiology and Immunology Laboratory of South China Agricultural University). After completing the challenge procedure, piglets were continuously observed for two weeks. The appearance of characteristic clinical symptoms such as physical appearance and mental state of piglets, as well as the occurrence of disease and death, were used as criteria for judging the protective effect, and relevant records were kept.

[0156] II. Results and Analysis

[0157] 1. Detection of PRRSV and PCV2-specific antibodies in serum of immunized piglets

[0158] Using the vaccines listed in Table 2, according to Figure 1 Piglets were immunized according to an immunization program. Peripheral blood was collected from piglets on days 0, 14, and 28 post-immunization, and serum was separated. The levels of PRRSV and PCV2 specific antibodies in different vaccine groups were detected to evaluate the immunization efficacy of the vaccines. When analyzing the data, absorbance (OD value) was measured at 450 nm using a UV spectrophotometer, and the threshold was calculated using the formula SP = (ODS - ODNC) / (ODPC - ODNC). An SP value greater than or equal to 0.16 was considered positive; an SP value less than 0.16 was considered negative. Statistical results of PRRSV specific antibody levels in peripheral serum of piglets from different vaccine groups showed (…). Figure 12 At 14 days after the first immunization, all pigs except the DMEM group showed positive PRRSV-specific antibody levels, with a significant increase. Furthermore, the PRRSV-specific antibody levels in the rAd-GP5+M+TBCap vaccine group were comparable to those in the commercial vaccine group, with the rAd-GP2+GP3+GP4+N+rAd-GP5+M+TBCap vaccine group showing relatively higher levels. After the second immunization, except for the DMEM group, the PRRSV-specific antibody levels in all other groups increased to varying degrees, with no significant difference among the three vaccine groups. These results indicate that the vaccines of this invention can stimulate piglets to produce high levels of PRRSV-specific antibodies and have a strong and sustained ability to induce antibody production.

[0159] The statistical results of the PCV2-specific antibody levels in the peripheral serum of piglets in the same vaccine group are shown below. Figure 12 At 14 days after the first immunization, all pigs except the DMEM group showed positive PCV2-specific antibody levels, with a significant increase. Furthermore, the PCV2-specific antibody levels in the rAd-GP5+M+TBCap vaccine group were comparable to those in the commercial vaccine group, and the levels in the rAd-GP2+GP3+GP4+N+rAdEasy-GP5+M+TBCap vaccine group were significantly higher than those in the rAd-GP5+M+TBCap vaccine group and the commercial vaccine group. After the second immunization, the PCV2-specific antibody levels in all pigs except the DMEM group continued to increase. At 28 days, there was no significant difference in PCV2-specific antibody levels among the rAd-GP5+M+TBCap vaccine group, the rAd-GP2+GP3+GP4+N+rAd-GP5+M+TBCap vaccine group, and the commercial vaccine group. These results indicate that the vaccines of this invention can stimulate piglets to produce high levels of PCV2-specific antibodies and have a strong ability to continuously induce an increase in antibody levels.

[0160] 2. Detection of PRRSV and PCV2 neutralizing antibodies in serum of immunized piglets

[0161] To verify the protective effect induced by the vaccine developed in this invention in piglets, serum samples were collected at 21, 28, and 35 days after the first immunization to detect the neutralizing antibody titer. The results are as follows: Figure 13 The results showed that the neutralizing antibody levels in piglets in the DMEM control group remained stable without significant changes. However, the neutralizing antibody levels in the commercial vaccine group and other experimental vaccine groups increased to varying degrees, all higher than those in the DMEM group. This indicates that both the rAd-GP2+GP3+GP4+N+rAd-GP5+M+TBCap vaccine group and the rAd-GP5+M+TBCap vaccine group can induce protective effects against PRRSV in piglets.

[0162] To verify the protective effect induced by the vaccine developed in this experiment in piglets, serum samples were collected at 21, 28, and 35 days after the first immunization to detect the neutralizing antibody titer. The results are as follows: Figure 13 In the DMEM control group, the neutralizing antibody levels in piglets remained stable without significant change. However, the neutralizing antibody levels in the commercial vaccine group and other experimental vaccine groups increased to varying degrees, all exceeding those in the DMEM group. This demonstrates that both the rAd-GP2+GP3+GP4+N+rAd-GP5+M+TBCap vaccine group and the rAd-GP5+M+TBCap vaccine group can induce protection against PCV2 in piglets.

[0163] 3. Detection of peripheral blood cytokines in immunized piglets

[0164] Depend on Figure 14As can be seen, the IFN-γ secretion levels in each vaccine group were significantly higher than those in the DMEM group (P<0.01). The levels in the rAdEasy-GP5+M+TBCap vaccine group were lower than those in the rAd-GP2+GP3+GP4+N+rAdEasy-GP5+M+TBCap vaccine group, indicating that these two recombinant adenovirus vector vaccines can induce IFN-γ secretion and trigger a Th1-type cellular immune response. Figure 14 b. As can be seen, the serum IL-2 secretion levels in all immunized piglet groups were significantly higher than those in the DMEM group (P < 0.05). The IL-2 secretion levels in the rAd-GP2+GP3+GP4+N+rAd-GP5+M+TBCap vaccine group, the rAd-GP5+M+TBCap vaccine group, and the commercially available vaccine group were comparable, indicating that the recombinant adenovirus vaccine prepared in this invention can increase the concentration of IL-2 in the body, promote the Th1 immune response, and induce cellular immunity. Figure 14 c. As can be seen, the IL-4 secretion levels in all vaccine groups were significantly higher than those in the DMEM group (P < 0.01), with the rAd-GP2+GP3+GP4+N+rAd-GP5+M+TBCap vaccine group showing the highest levels. The recombinant adenovirus vector vaccine group also showed higher levels than the commercially available vaccine group. This indicates that the recombinant adenovirus vaccine developed in this experiment can stimulate the body to secrete IL-4 and induce a Th2-type cellular immune response. In summary, the serum cytokine secretion levels of piglets 35 days after the first immunization, detected by ELISA, showed that the secretion levels of IFN-γ, IL-2, and IL-4 in all vaccine groups were higher than those in the DMEM group after immunization. This indicates that all experimental groups can stimulate the production of Th1 and Th2-type cytokines and induce Th1 and Th2-type cellular immune responses.

[0165] 4. Detection of peripheral blood lymphocyte proliferation activity in immunized piglets

[0166] PBMC proliferation and activation levels are important indicators for assessing the body's immune response and can reflect the overall immune level. PBMCs contain a large number of lymphocytes and some monocytes, with T lymphocytes being the predominant type. To evaluate the effect of vaccines on the immune response of experimental pigs, this invention used the CCK8 kit to treat PBMCs from each group of experimental pigs isolated on day 35 after the initial immunization and then cultured them in vitro. After stimulation with inactivated PRRSV and PCV2 (NADC30-like PRRSV strain and PCV2 d strain) for 72 h, the cells were treated with the reagents in the CCK8 kit and cultured for another 4 h. The OD values ​​were measured using a UV spectrophotometer to calculate the lymphocyte SI.

[0167] The results show that, by Figure 15As shown in Figure a, compared with the DMEM group, the SI values ​​of all vaccine groups were significantly improved. Among them, the SI values ​​of the rAd-GP2+GP3+GP4+N+rAd-GP5+M+TBCap vaccine group and the rAd-GP5+M+TBCap vaccine group were comparable, and the SI values ​​of these two groups were higher than those of the commercially available vaccine group. This indicates that the vaccine developed in this invention can significantly enhance the immune response of piglet PBMCs to PRRSV, stimulate PBMC proliferation activity, and resist viral infection. Figure 15 As shown in b, compared with the DMEM group, all vaccine groups also showed significant increases in SI values ​​to varying degrees, and the levels in the rAd-GP2+GP3+GP4+N+rAd-GP5+M+TBCap vaccine group, the rAd-GP5+M+TBCap vaccine group, and the commercial vaccine group were comparable. This indicates that the vaccines in this study can significantly enhance the immune response of piglet PBMCs to PCV2, stimulate PBMC proliferation, and enhance the body's resistance to the virus.

[0168] 6. PRRSV challenge protection test

[0169] To test the protective effect of the vaccine against PRRSV challenge, 10 6 TCID 50 Pigs were challenged with intramuscular injection of NADC30-like strain PRRSV at a concentration of / mL. Following challenge, piglets experienced stunted growth and development, showing a significant difference compared to healthy piglets in the same litter. Figure 16 Compared to the second row, these piglets show decreased appetite, significant weight loss, and rough, dull coats. They are lethargic, drowsy, and unwilling to move, often huddling together or hiding in corners. Some exhibit rapid breathing, a significantly increased respiratory rate, diarrhea, and may show a pain response when touched. Figure 16 (First row). In addition, some pigs died ( Figure 16 (Third row). Piglet survival rate after viral challenge is as follows: Figure 17 As shown, one pig in the rAd-GP2+GP3+GP4+N+rAd-GP5+M+TBCap vaccine group died on day 6 after challenge; one pig in the rAd-GP5+M+TBCap vaccine group died on days 3 and 9 after challenge; one pig in the commercial vaccine group died on day 11 after challenge; and one pig in the DMEM control group died on days 1, 4, and 14 after challenge. The results indicate that the recombinant adenovirus vaccines expressed in this invention can all provide some degree of protection against PRRSV NADC30-like strain infection.

[0170] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A bivalent vaccine of porcine PRRSV and PCV2 recombinant adenovirus, characterized in that... Recombinant adenovirus 1 expressing PRRSV GP5, M and PCV2TBCap antigen proteins and recombinant adenovirus 2 expressing PRRSV GP2, GP3, GP4 and N antigen proteins; The PRRSV GP5, M and PCV2 TBCap antigen proteins comprise P5+M(HP)+2A+GP5+M(CH-1a) fusion protein and GP5+M(NAD30)+2A+TBCap fusion protein, the amino acids of which are shown in SEQ ID NO:1 and 2, respectively. The PRRSV GP2, GP3, GP4 and N antigen proteins include GP3+2A+GP4 fusion protein and PG2+2A+N fusion protein, whose amino acid sequences are shown in SEQ ID NO:3 and 4, respectively.

2. The porcine PRRSV and PCV2 recombinant adenovirus bivalent vaccine according to claim 1, characterized in that: The nucleotide sequence encoding the P5+M(HP)+2A+GP5+M(CH-1a) fusion protein is shown in SEQ ID NO:5; The nucleotide sequence encoding the GP5+M(NAD30)+2A+TBCap fusion protein is shown in SEQ ID NO:6; The nucleotide sequence encoding the GP3+2A+GP4 fusion protein is shown in SEQ ID NO:7; The nucleotide sequence encoding the PG2+2A+N fusion protein is shown in SEQ ID NO:

8.

3. The porcine PRRSV and PCV2 recombinant adenovirus bivalent vaccine according to claim 1, characterized in that: In the vaccine, the volume ratio of recombinant adenovirus 1 to recombinant adenovirus 2 is 1:

1.

4. The porcine PRRSV and PCV2 recombinant adenovirus bivalent vaccine according to claim 1, characterized in that: The dosage concentration of recombinant adenovirus 1 and recombinant adenovirus 2 in the vaccine is 1.0 × 10⁻⁶. 8 TCID 50 / mL.

5. The method for preparing the porcine PRRSV and PCV2 recombinant adenovirus bivalent vaccine according to any one of claims 1-4, characterized in that... It includes the following steps: (1) Insert the nucleotide sequence encoding the GP5+M(HP)+2A+GP5+M(CH-1a) fusion protein and the nucleotide sequence encoding the GP5+M(NAD30)+2A+TBCap fusion protein into an adenovirus shuttle plasmid containing dual promoters, wherein the GP5+M(HP)+2A+GP5+M(CH-1a) fusion protein and the GP5+M(NAD30)+2A+TBCap fusion protein are each expressed using one promoter, thus obtaining adenovirus shuttle plasmid 1; (2) Insert the nucleotide sequence encoding the GP3+2A+GP4 fusion protein and the nucleotide sequence encoding the PG2+2A+N fusion protein into an adenovirus shuttle plasmid containing dual promoters, wherein the GP3+2A+GP4 fusion protein and the PG2+2A+N fusion protein are each expressed using one promoter, to obtain adenovirus shuttle plasmid 2. (3) After linearizing adenovirus shuttle plasmid 1 and adenovirus shuttle plasmid 2, they were co-transformed with adenovirus backbone vector into competent cells of adenovirus system to obtain recombinant adenovirus plasmid. (4) The recombinant adenovirus plasmid was packaged to obtain recombinant adenovirus 1 expressing PRRSV GP5, M and PCV2 TBCap antigen proteins and recombinant adenovirus 2 expressing PRRSV GP2, GP3, GP4 and N antigen proteins, respectively. (5) The recombinant adenovirus 1 expressing PRRSV GP5, M and PCV2 TBCap antigen proteins obtained in step (4) and the recombinant adenovirus 2 expressing PRRSVGP2, GP3, GP4 and N antigen proteins are mixed to obtain a porcine PRRSV and PCV2 recombinant adenovirus bivalent vaccine.

6. The method for preparing the porcine PRRSV and PCV2 recombinant adenovirus bivalent vaccine according to claim 5, characterized in that: The adenovirus shuttle plasmid mentioned in step (1) is pAdTrack.

7. The method for preparing the porcine PRRSV and PCV2 recombinant adenovirus bivalent vaccine according to claim 5, characterized in that: The adenovirus backbone vector mentioned in step (3) is pAdEasy-1.

8. The method for preparing the porcine PRRSV and PCV2 recombinant adenovirus bivalent vaccine according to claim 5, characterized in that: The competent cells mentioned in step (3) are BJ-5183 competent cells.

9. The method for preparing the porcine PRRSV and PCV2 recombinant adenovirus bivalent vaccine according to claim 5, characterized in that: The host cells used for packaging in step (4) are HEK 293 cells.

10. The use of the porcine PRRSV and PCV2 recombinant adenovirus bivalent vaccine according to any one of claims 1-4 in the preparation of products for the prevention and treatment of porcine PRRSV or PCV2.

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