A recombinant PRRSV-GP3-GP5-M+PCV2-TBCap protein, a vaccine, and its applications.

Abstract

This invention relates to the field of biotechnology, and particularly to a PRRSV-GP3-GP5-M+PCV2-TBCap recombinant protein, a vaccine, and its applications. This invention successfully expresses PRRSV GP5, GP3, and M proteins and PCV2TBCap protein using a cyclovir expression system. This recombinant protein can induce a favorable humoral and cellular immune response in the body, exhibiting good immunogenicity and safety.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a recombinant protein PRRSV-GP3-GP5-M+PCV2-TBCap, a vaccine, and its applications. Background Technology

[0002] Currently, there are no commercially available bivalent vaccines for porcine reproductive and respiratory syndrome (PRRS) and porcine circovirus type 2 (PCV2), but related research has never stopped. Using the encoding genes of the GP3 and GP5 proteins of PRRSV (which have good immunogenicity) and the Cap protein of PCV2, research has constructed a bivalent nucleic acid vaccine for PRRS and PCV2, laying the research foundation for this vaccine. Simultaneously, there are also inactivated bivalent vaccines for PCVAD and PRRS developed using isolated virulent strains of PCV2 and PRRSV. Furthermore, researchers have invented a method for preparing a bivalent vaccine by freeze-drying a combination of live PRRS virus (R98 strain) and inactivated PCV2 virus (SH strain), which provides good protection for immunized pigs in challenge protection tests. Currently, there are monovalent vaccines for PRRS and PCVAD on the market, but inconsistent immunization schedules require multiple injections, which can easily cause stress in animals and increase workload. Although multivalent vaccines are relatively convenient, some immunogenic components can interfere with each other, affecting the immunization effect. Therefore, the technical basis for developing a bivalent vaccine using baculovirus expression systems to express immunogenic proteins of PRRSV and PCV2 can be considered feasible.

[0003] Baculovirus expression vector systems (BEVS) are expression systems that use baculoviruses as vectors to insert foreign genes for efficient expression of foreign proteins. Due to the infection specificity of baculoviruses and their non-pathogenicity in mammals, they are widely used. Mammalian expression systems offer advantages such as accurate modification and folding of expressed proteins, and expression in transgenic form, but their expression speed is slow, and their cost is high, making them unsuitable for large-scale production. Yeast expression systems can also modify expressed proteins and can be expressed through yeast secretion, with a relatively lower cost compared to mammalian expression systems. However, the glycosylation of proteins expressed by yeast expression systems differs from that of natural proteins, and the expression efficiency is lower, which limits the application scope of this system. Compared to the eukaryotic expression systems mentioned above, BEVS is a more balanced protein expression system. Its main advantages include: a smaller baculovirus genome, more restriction endonuclease sites, a larger capacity for foreign genes, and ease of operation; post-expression modification of the target protein, resulting in a structure and properties closer to the natural protein and better biological activity; and high safety, as baculoviruses do not infect humans or other mammals. Insect cell lines are easy to grow, and suspension culture can efficiently express large amounts of exogenous genes at a low cost.

[0004] Currently, PRRS and PCVAD have become two major infectious diseases affecting the development of my country's pig industry. Epidemiological surveys show that mixed infections of the two pathogens are common in PCVAD cases, leading to immunosuppression and decreased immunity in pigs, making them susceptible to secondary infections by various viruses or bacteria. This severely impacts the production performance of pig herds and causes significant economic losses to the pig industry. At present, disease control still relies primarily on single-drug immunization against these two diseases. However, repeated immunizations cause adverse stress and interference between different antigenic components, both of which affect the effectiveness of immunization. Therefore, simplifying immunization programs and developing safer and more effective bivalent vaccines are of great significance for the prevention and control of the occurrence and spread of PCVAD and PRRS. 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 recombinant protein PRRSV-GP3-GP5-M+PCV2-TBCap.

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

[0007] Another object of the present invention is to provide applications of the above-mentioned recombinant protein.

[0008] A fourth object of the present invention is to provide a vaccine comprising the above-described recombinant protein.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A PRRSV-GP3-GP5-M+PCV2-TBCap recombinant protein comprising recombinant GP5 protein, recombinant GP3 ​​protein, recombinant TBCap protein, and recombinant M protein, the amino acid sequence of which is shown in SEQ ID No: 1-4;

[0011] The nucleotide sequences of the recombinant GP5 protein, recombinant GP3 ​​protein, recombinant TBCap protein, and recombinant M protein are shown in SEQ ID No: 5-8;

[0012] A multi-gene expression cassette comprising genes encoding the aforementioned PRRSV-GP3-GP5-M+PCV2-TBCap recombinant protein, wherein the genes encoding recombinant GP5 protein and recombinant GP3 ​​protein are respectively linked to a polyhedrin (polh) promoter; the genes encoding recombinant TBCap protein and recombinant M protein are linked to a p10 promoter; and the polyhedrin promoter and p10 promoter are in opposite directions.

[0013] A recombinant transfer plasmid containing the above-mentioned multi-gene expression cassette was prepared by the following method:

[0014] (1) The gene sequence encoding recombinant TBCap protein containing Nhe I and Xho I restriction sites and the recombinant transfer plasmid pFBDM-GP5 were digested with Nhe I and Xho I respectively and then ligated to obtain pFBDM-GP5-TBCap; the recombinant transfer plasmid pFBDM-GP5 was obtained by ligating the gene encoding recombinant GP5 protein with pFBDM.

[0015] (2) The gene sequence encoding the recombinant M protein containing Nhe I and Xho I restriction sites and the recombinant transfer plasmid pFBDM-GP3 were digested with Nhe I and Xho I respectively and then ligated to obtain pFBDM-GP3-M; the group transfer plasmid pFBDM-GP3 was obtained by ligating the gene encoding the recombinant GP3 ​​protein with pFBDM.

[0016] (3) pFBDM-GP5-TBCap was digested with restriction endonucleases PmeI / AvrII to obtain expression cassettes of GP5 and TBCap genes; pFBDM-GP3-M was cleaved with restriction endonucleases BstZ17I / SpeI.

[0017] (4) The expression cassettes of the obtained GP5 and TBCap genes were ligated with the enzyme-digested pFBDM-GP3-M to obtain the recombinant transfer plasmid pFBDM-GP3-GP5-M-TBCap;

[0018] A recombinant baculovirus plasmid was obtained by transforming DH10Multibac competent cells with the above-mentioned recombinant transfer plasmid pFBDM-GP3-GP5-M-TBCap.

[0019] A recombinant baculovirus was obtained by transfecting sf9 insect cells with the above-mentioned recombinant baculovirus plasmid rBac-TBCap-GP3-GP5-M, followed by isolation and purification.

[0020] The method for preparing the PRRSV-GP3-GP5-M+PCV2-TBCap recombinant protein includes the following steps:

[0021] The above recombinant baculovirus was transfected into High-five cells. After transfection, the cells were centrifuged and the supernatant was discarded. The cells were resuspended and lysed, centrifuged, and the supernatant was collected to obtain the PRRSV-GP3-GP5-M+PCV2-TBCap recombinant protein.

[0022] The preferred MOI for transfection is 5.0, and the preferred transfection time is 72–96 h.

[0023] The application of the PRRSV-GP3-GP5-M+PCV2-TBCap recombinant protein in the preparation of a bivalent vaccine against porcine reproductive and respiratory syndrome and porcine circovirus type 2;

[0024] A bivalent vaccine against porcine reproductive and respiratory syndrome and porcine circovirus type 2 contains the above-mentioned PRRSV-GP3-GP5-M+PCV2-TBCap recombinant protein;

[0025] The vaccine preferably also contains ISA201VG adjuvant;

[0026] The method for preparing the vaccine includes the following steps:

[0027] The PRRSV-GP3-GP5-M+PCV2-TBCap recombinant protein was mixed with an adjuvant to obtain the vaccine;

[0028] The preferred mass ratio of the PRRSV-GP3-GP5-M+PCV2-TBCap recombinant protein to the adjuvant is 1:1;

[0029] The principle of this invention:

[0030] Based on the NADC30-Like ORF3 / ORF6 sequence (accession number: JN654459.1) included in GenBank, a base sequence encoding a 6×His tag protein was added before the terminator. After optimization based on insect cell codon preference, the gene sequences of recombinant GP3 ​​and M proteins were obtained.

[0031] The bait epitopes (27–31 aa) in the NADC30-Like ORF5 gene (accession number: JN654459.1) sequence were replaced with neutralizing epitopes (37–45 aa), the asparagine triplets (AAC) at positions N34, N44, and N51 of the ORF5-based cDNA were replaced with alanine triplets (GCC), and a base sequence encoding a 6×His tag protein was added before the terminator. After optimization for insect cell codon preference, the gene sequence of the recombinant GP5 protein was obtained.

[0032] The basic gene sequence of the PCV2-TBCap protein was selected from the ORF2 gene of PCV2LG strain (HM038034.1) in GenBank. T and B cell antigenic epitopes and the base sequence of a 6×His tag protein were introduced into the ORF2 gene, finally obtaining the gene sequence of the recombinant protein PCV2-TBCap. This recombinant protein gene sequence was then synthesized by a biotechnology company. High-titer recombinant baculoviruses Ac-TBCap-2, Ac-GP3-GP5-M, and Ac-GP3-GP5-M-TBCap were constructed in sf9 cells. These were then seeded into High Five cells, which have high expression efficiency, to optimize the expression conditions of the target protein.

[0033] To evaluate the immunogenicity of the recombinant proteins GP3-GP5-M, TBCap, and GP3-GP5-M-TBCap, the three expressed recombinant proteins were mixed with ISA201VG adjuvant at a 1:1 mass ratio and emulsified separately to prepare vaccines. Fifteen 4-week-old PRRSV / PCV2 antigen- and antibody-negative piglets were divided into five groups (3 piglets / group) and immunized with PRRSV-GP3-GP5-M vaccine, PCV2-TBCap vaccine, PRRSV-GP3-GP5-M+PCV2-TBCap vaccine, PRRSV+PCV2 commercial vaccine, and PBS, respectively. A second immunization was administered on day 14 after the initial immunization. Blood samples were collected on days 0, 7, 14, 21, 28, and 35 after the initial immunization to assess the immunization efficacy. The safety of the three prepared vaccines was evaluated. Clinical and histopathological observations revealed no adverse reactions or pathological changes, indicating that the prepared vaccines were safe.

[0034] The present invention has the following advantages and effects compared with the prior art:

[0035] This study successfully expressed PRRSV GP5, GP3, and M proteins and PCV2 TBCap protein using a modified baculovirus expression system. The results demonstrated that these four recombinant proteins can induce favorable humoral and cellular immune responses, exhibiting good immunogenicity and safety. This research lays the foundation for further development of a PRRSV+PCV2 bivalent vaccine. Attached Figure Description

[0036] Figure 1 This is a spectrum of the transfer vector pFBDM.

[0037] Figure 2 These are the spectra of the recombinant transfer plasmids pFBDM-GP3, pFBDM-GP5, pFBDM-M, and pFBDM-TBcap, where a: pFBDM-GP3; b: pFBDM-GP5; c: pFBDM-M; d: pFBDM-TBcap.

[0038] Figure 3 This is a schematic diagram of assembling a multi-gene expression cassette.

[0039] Figure 4 These are the spectra of the recombinant transfer plasmids pFBDM-GP3-GP5-M-TBCap and pFBDM-GP3-GP5-M, where a: pFBDM-GP3-GP5-M-TBCap; b: pFBDM-GP3-GP5-M.

[0040] Figure 5 The results of indirect immunofluorescence detection (40×) of the target protein are shown in the figure. Among them, a, c, e, g: Ac-TBCap, Ac-GP3-GP5-M, Ac-TBCap-GP3-GP5-M and wild-type baculovirus group under white light; b, d, f, h: Ac-TBCap, Ac-GP3-GP5-M, Ac-TBCap-GP3-GP5-M and wild-type baculovirus group under fluorescence.

[0041] Figure 6 The images show the results of Western blot analysis of the recombinant protein. In a: 1: High-five cells infected with wild-type baculovirus; 2: High-five cells infected with Ac-TBCap; b: 1: High-five cells infected with wild-type baculovirus; 2: High-five cells infected with Ac-GP3-GP5-M; c: 1: High-five cells infected with Ac-GP3-GP5-M-TBCap; 2: High-five cells infected with wild-type baculovirus.

[0042] Figure 7 This is a diagram showing the results of exploring the expression conditions for recombinant proteins.

[0043] Figure 8 The graph shows the results of recombinant protein expression level determination. a: Expression level determination of single-copy recombinant TBCap protein, 1-5: 1000, 500, 250, 125, 62.5 μg / mL, respectively; 6: Ac-TBCap 5-fold concentrated cell lysate; b: Expression level determination of GP3-GP5-M recombinant protein, 1-6: 1000, 500, 250, 125, 62.5, 31.25 μg / mL, respectively; 7: Ac-GP3-GP5-M 5-fold concentrated cell lysate; c: Expression level determination of GP3-GP5-M-TBCAP recombinant protein, 1-5: 1000, 500, 250, 125, 62.5 μg / mL, respectively; 6: Ac-GP3-GP5-M-TBCAP 5-fold concentrated cell lysate.

[0044] Figure 9This is a graph showing the results of ELISA detection of PRRSV-specific antibodies in piglet serum. An asterisk (*) indicates a significant difference between groups (p<0.05), *** indicates p<0.05, *** indicates p<0.001, **** indicates p<0.0001; ns indicates no significant difference between groups (p>0.05).

[0045] Figure 10 This is a graph showing the results of ELISA detection of PCV2-specific antibodies in piglet serum. Asterisks indicate significant differences between groups (p<0.05), where **: p<0.01, ***: p<0.001, ****: p<0.0001; ns: no significant difference between groups (p>0.05).

[0046] Figure 11 This is a graph showing the results of the analysis of the titers of anti-PRRSV and anti-PCV2 neutralizing antibodies in peripheral blood of piglets. In the graph, asterisks represent significant differences between groups (p<0.05), ***: p<0.0001; ns: no significant difference (p>0.05).

[0047] Figure 12 This is an analysis of the results of detecting IL-2, IFN-γ, and IL-4 in the supernatant of peripheral blood lymphocytes from piglets. In the figure, asterisks represent significant differences between each vaccine group and the PBS group (p<0.05), *: p<0.05, **: p<0.01, ***: p<0.001; ns: no significant difference between groups (p>0.05). Detailed Implementation

[0048] 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.

[0049] Example 1

[0050] I. Gene Sequence Design and Synthesis of Recombinant Proteins

[0051] 1. Design of gene sequences for recombinant GP3 ​​and recombinant M proteins

[0052] Based on the NADC30-Like ORF3 / ORF6 sequence (accession number: JN654459.1) indexed in GenBank, a base sequence encoding a 6×His tag protein was added before the terminator. After codon preference optimization in insect cells, the gene sequences of recombinant GP3 ​​protein and recombinant M protein were obtained, and their nucleotide sequences are shown below:

[0053] Nucleotide sequence of recombinant GP3 ​​protein:

[0054] Amino acid sequence of recombinant GP3 protein:

[0055] MVNSCALLHIFLCCSFLHFICCAMATSANSTYCFWFPLVRGNFSFELMVNYTVCPPCLTRQAAAQIFEPGRSLWCRIGHDRCEESDHDELEFAVPPGLSNESHLTSVYAWLAFLSFSYTAQFHPEIFGIGNVSKVYVDIQKQLICAVHDGQNATLPSHDNITAVYQTYYQHQVDGGNWFHLEWLRPFFSSWLVLNVSWFLRRSPVSRVSVRVFQTLRPTPPAQQVPLSSKTSAVLGIATRPLRRFAKSLNAARRHHHHHH.

[0056] Nucleotide sequence of recombinant M protein:

[0057] ATGGGCAGCAGCATCGACGACTTTTGCAACGATAGCACCGCCGTGCAGAAAGTGCTGCTGGCCTTCAGCATCACCTACA

[0058] CCCCCATCATGATTTACGCCCTGAAGGTGTCCCGCGGACGATTGCTGGGCCTGCTGCACCTGCTGATCTTCCTGAATTGC

[0059] GCCTTCACCTTCGGCTACATGACCTTCGTGCACTTCCAAAGCACCAACAAGGTGGCTTTGACAATGGGAGCGGTGGTGG

[0060] CTCTGTTGTGGGGAGTGTACTCCGCTATGGAGACTTGGCGCTTCATTACCAGCCGTTGCCGCCTGTGCCTGTTGGGCCGC

[0061] AAGTACATCCTGGCTCCAGCTCATCACGTGGAAAGTGCGGCTGGCTTCCACCCAATCACCGCCAGCGACAATCATGCTT

[0062] TCGTGGTGCGCCGTCCAGGAAGTACAACAGTGAACGGCACCCTGGTGCCAGGATTCAAGAGTCTGGTGCTGGGAGGAC

[0063] GCCGAGCCGTGAAACGAGGAGTGGTGAACCTGGTGAAGTACGCCAAGCACCACCACCACCACCACTAG

[0064] Amino acid sequence of recombinant M protein:

[0065] MGSSIDDFCNDSTAVQKVLLAFSITYTPIMIYALKVSRGRLLGLLHLLIFLNCAFFTFGYMTFVHFQSTNKVALTMGAVVA LLWGVYSAMETWRFITSRLCLLGRKYILAPAHHVESAAGFHPITASDNHAFVVRRPGSTTVNGTLVPGFKSLVLGGRRAV KRGVVNLVKYAKHHHHHH.

[0066] 2. Design of the gene sequence for recombinant GP5 protein

[0067] The bait epitopes (27–31 aa) in the NADC30-Like ORF5 gene sequence (accession number: JN654459.1) were replaced with neutralizing epitopes (37–45 aa), and the asparagine triplets (AAC) at positions N34, N44, and N51 of the ORF5 gene cDNA were replaced with alanine triplets (GCC). A base sequence encoding a 6×His tag protein was added before the terminator. After codon preference optimization in insect cells, the gene sequence of the recombinant GP5 protein was obtained, and its nucleotide sequences are shown below:

[0068] Nucleotide sequence of recombinant GP5 protein:

[0069] ATGCTGGGCAAGTGCCTGACCGCCGGATACTGCAGCCAGCTGCCCTTCCTGTGGTGCATTGTGCCCTTTTGCTTCGCCAGCCATCTGCAGCTGATCTACGCCCTGAATAGCGCCAGCAGCAGCCATCTGCAGCTGATCTACGCCCTGACCATTTGCGAGCTGGCGGGAACCGATTGGCTGAACGAGCGCTTCTATTGGGCCGTGGAGACCTTCGTGATCTTCCCCGTGCTGACCCACATCGTGAGCTACGGCGCCCTGACCACCAGCCATTTCCTGGACACCGTGGGACTGATCACCGTGTCCACAGCCGGATACTACCATCGCCGCTACGTGCTGAGCAGCATTTACGCCGTGTGCGCTCTGGCCGCTCTGATTTGCTTTGCCATCCGCCTGGCCAAGAATTGCATGAGTTGGCGCTACAGTTGCACCCGCTACACCAACTTCCTGCTGGACACCAAGGGCAAGCTGTACCGTTGGCGTAGCCCCGTGATCATCGAGAAGGGAGGCAAGGTGGACGTGGGAGGACACCTGATCGATCTGAAGCGCGTCGTGTTGGATGGTAGTGCCGCTACCCCAGTGACCAAGATTAGCGCCGAGCAGTGGGGACGCCCACATCATCATCACCACCACTAA

[0070] Amino acid sequence of recombinant GP5 protein:

[0071] MLGKCLTAGYCSQLPFLWCIVPFCFASHLQLIYALNSASSSHLQLIYALTICELAGTDWLNERFYWAVETFVIFPVLTHIVS YGALTTSHFLDTVGLITVSTAGYYHRRYVLSSIYAVCALAALICFAIRLAKNCMSWRYSCTRYTNFLLDTKGKLYRWRSPVII EKGGKVDVGGHLIDLKRVVLDGSAATPVTKISAEQWGRPHHHHHH.

[0072] 3. Gene sequence of recombinant protein PCV2-TBCap

[0073] The basic gene sequence of the PCV2-TBCap protein was selected from the ORF2 gene of PCV2LG strain (HM038034.1) in GenBank. T and B cell antigenic epitopes and the base sequence of a 6×His tag protein were introduced into the ORF2 gene, finally obtaining the recombinant protein PCV2-TBCap gene sequence, the nucleotide sequences of which are shown below:

[0074] Nucleotide sequence of recombinant protein PCV2-TBCap:

[0075]

[0076] The amino acid sequence of recombinant protein PCV2-TBCap:

[0077] MKFLVNVALVFMVVYISYIYACHIEKAKGTDQQNKEYCSKEGGKWWDGYHGEEVVVIDDFYGWGGTVRTPSWAVDMMRFNINDFVPPGGGGGQGDRGVGSTAVILDDNFVTGGSTIDYFQPNNKRGGNVDHVGLGIAFGGTYPRRRFRRRRHRPRSHLGLILRRRPWLVHPRHRYRWRRKNGIFNTRLS CTFGYTVKATTVRTPSWAVDMMRFNINDFVPPGGGTNEISIPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVILDDNFVTRATALTYGPYVNYSSRHTIPQPFSYHSRYFTPKPVLDSTIDYFQPNNKRNQLWLRLQTSANVDHVGLGIAFENSTYDQDYNIRVTMYVQFREFNLKDPPLHHHHHH.

[0078] The above recombinant protein gene sequences were given to a biotechnology company for synthesis.

[0079] II. Construction of Recombinant Transfer Plasmids

[0080] 1. Primer design

[0081] Primers were designed based on the optimized gene sequence using Primer 5.0 software (Table 1). BamH I restriction sites were introduced into the GP3-ph-P1, GP5-ph-P1, M-ph-P1 and TBCap-ph-P1 primers; Hind III restriction sites were introduced into the GP3-ph-P2, GP5-ph-P2 and M-ph-P2 primers; Sac I restriction site was introduced into the TBCap-ph-P2 primer; Nhe I restriction site was introduced into the TBCap-p10-P1 primer; and Xho I restriction site was introduced into the TBCap-p10-P2 primer.

[0082] Table 1 Primer sequences

[0083]

[0084] 2. Using the synthesized GP3, GP5, M, and TBCap fragments as templates, and the primers listed in Table 1 as amplification primers, PCR was performed. The PCR reaction system was: Golden Star T6 Super PCR Mix (1.1×) 22.0 μL, Primer P1 1.0 μL, Primer P2 1.0 μL, and DNA template 1.0 μL. The PCR reaction program was: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 60 s, for a total of 30 cycles; and 72℃ extension for 5 min. The amplified target fragment was subjected to nucleic acid electrophoresis, and the target band was purified by gel extraction according to the Omega gel extraction kit instructions.

[0085] 3. The target fragment and pFBDM vector (Takara Bio Engineering (Dalian) Co., Ltd.) purified in step (2) were double-digested with the restriction endonucleases listed in Table 1 according to the following digestion system: 10×FastDigest Buffer 2.0 μL, FastDigest A 1.0 μL, FastDigest B 1.0 μL, PCR product / pFBDM 10.0 / 2.0 μL, and ddH2O to a final volume of 20.0 μL. The digestion system was prepared according to the restriction endonuclease used, and the reaction was carried out in a 37℃ water bath for 30 min, followed by termination of digestion at 85℃. After nucleic acid electrophoresis, the fragments were purified by gel extraction.

[0086] 4. The purified target fragment and pFBDM vector were ligated using the following ligation system: 4.0 μL 5×Ligase Reaction Buffer, 3.0 μL linear plasmid, 10.0 μL target fragment, 0.5 μL T4 DNA Ligase Enzymes, and ddH2O to a final volume of 20.0 μL. The reaction system was placed in a 16℃ metal bath for 12 h to obtain the corresponding ligation product.

[0087] 5. The ligation product obtained in step (4) was transformed according to the chemical transformation method of DH5α competent cells of Shanghai Weidi Biotechnology Co., Ltd., and cultured on LB agar plates (Amp resistant) for 12-16 h. Positive single colonies were picked and inoculated into 5 mL of LB liquid medium (Amp resistant), and cultured on a bacterial shaker (200 r / min) at 37℃ for 12-16 h before extracting the recombinant plasmid.

[0088] 6. The recombinant plasmids were identified using double enzyme digestion. The presence of bands matching the size of the target fragments GP3, GP5, M, and TBCap was considered a successful construction of the recombinant transfer plasmids. The successfully constructed recombinant plasmids were then sent to a biotechnology company for sequencing. The correctly identified recombinant transfer plasmids were named pFBDM-GP3, pFBDM-GP5, pFBDM-M, and pFBDM-TBcap, respectively. Figure 2 ).

[0089] 8. Construction of multi-gene expression cassettes

[0090] The pFBDM vector is a vector containing two promoters: the p10 promoter and the polyhedrin (polh) promoter. Figure 1 It is particularly suitable for generating multi-gene expression cassettes, utilizing single restriction sites of PmeI, AvrII, BstZ17I, and SpeI on the pFBDM vector, through... Figure 3 The demonstrated method involves enzyme digestion and ligation to obtain a multi-gene expression cassette. The specific method is as follows:

[0091] (1) The GP5 and TBCap genes were cloned into the MCS1 and MCS2 of pFBDM, respectively. The specific method was as follows: pFBDM-TBcap was used as a template, and the TBCap gene sequence containing Nhe I and Xho I restriction sites was amplified by primers TBCap-p10-P1 and TBCap-p10-P2. The gene sequence and the recombinant transfer plasmid pFBDM-GP5 obtained in step 6 were digested with Nhe I and Xho I respectively and then ligated to transform DH5α competent cells. Positive single colonies were picked, expanded cultured, and the recombinant plasmid was extracted and identified to obtain pFBDM-GP5-TBCap.

[0092] (2) The GP3 and M genes were introduced into the MCS1 and MCS2 of another pFBDM vector by cloning. The specific method is as follows: the M gene sequence containing Nhe I and Xho I restriction sites was amplified by primers M-p10-P1 and M-p10-P2. The gene sequence and the recombinant transfer plasmid pFBDM-GP3 obtained in step 6 were digested with Nhe I and Xho I respectively and ligated to transform DH5α competent cells. Positive single colonies were picked, expanded cultured, and the recombinant plasmid was extracted and identified to obtain pFBDM-GP3-M as module M.

[0093] (3) The entire expression cassette containing the GP5 and TBCap genes was digested and excised by restriction endonuclease (PmeI / AvrII). Then, the pFBDM vector containing the GP3 and M genes was cut from module M by restriction endonuclease (BstZ17I / SpeI). Since SpeI and AvrII can produce compatible sticky ends, while BstZ17I and PmeI can produce the same blunt ends, the expression cassette containing the GP5 and TBCap genes obtained by restriction endonuclease digestion was finally ligated to the pFBDM vector containing the GP3 and M genes to obtain the recombinant plasmid pFBDM-GP3-GP5-M-TBCap.

[0094] (4) Using the same method, recombinant plasmid pFBDM-GP3-GP5-M was obtained based on pFBDM-GP5 and pFBDM-GP3-M. Figure 4 ).

[0095] The recombinant plasmid pFBDM-GP3-GP5-M-TBCap expresses the recombinant protein TBCap-GP3-GP5-M via the promoters p10 and polyH, respectively.

[0096] The nucleotide sequences of TBCap and M guided by the first promoter (p10) are shown below:

[0097]

[0098]

[0099] The nucleotide sequences of GP3 and GP5 guided by the second promoter (polh) are shown below:

[0100]

[0101]

[0102] Note: The underlined part represents GP3, the wavy part represents GP5, the double-strikeed part represents M, and the dotted underlined part represents the nucleotide sequence of the GP3-GP5-M recombinant protein expressed by the TBCap recombinant plasmid pFBDM-GP3-GP5-M, as shown below:

[0103]

[0104]

[0105] Note: The underlined part is GP3, the wavy part is GP5, and the dotted underline part is TBCap.

[0106] Example 2

[0107] I. Obtaining Recombinant Baculovirus Plasmids

[0108] 1. Transformation of recombinant baculovirus plasmids

[0109] DH10Multibac competent cells were prepared using conventional methods. The recombinant transfer plasmids pFBDM-TBCap, pFBDM-GP3-GP5-M, and pFBDM-TBCap-GP3-GP5-M, successfully identified in Example 1, were then transformed into DH10Multibac competent cells. The specific methods are as follows:

[0110] (1) Add 1 ng of recombinant transfer plasmid to 100 μL of DH10 Multibac competent cells, mix gently, and incubate on ice for 25 min. After the ice bath, heat shock at 42℃ for 45 s, and then incubate on ice again for 2 min. Add 900 μL of SOC medium and culture at 37℃ and 200 r / min for 4 h.

[0111] (2) Take an appropriate amount of the transformed bacteria from step (1) and spread it on LB (Gen, Kan, Amp resistant) solid medium treated with IPTG and X-Gel. Place the plate in a 37℃ bacterial incubator and invert it for 48 hours. Observe the colony color.

[0112] (3) The white colonies in step (2) were streaked and purified on LB (Gen, Kan, Amp resistant) solid medium treated with IPTG and X-Gel, and cultured in a bacterial incubator at 37°C for 48 hours.

[0113] 2. Extraction of recombinant baculovirus plasmid

[0114] White single colonies were picked and inoculated into 5 mL of SOC liquid medium (Amp, Kan, and Gen resistant) and cultured at 37°C on a shaker at 200 rpm for 24–36 h. Recombinant baculovirus plasmids were extracted using standard methods and identified by PCR (using universal primers M13-F and M13-R) to confirm successful insertion of the corresponding target gene into the baculovirus plasmids, yielding recombinant baculovirus plasmids rBac-TBCap, rBac-GP3-GP5-M, and rBac-TBCap-GP3-GP5-M. The concentrations were determined, and the plasmids were stored at 4°C for later use.

[0115] II. Obtaining Recombinant Baculovirus

[0116] 1. Transfection of sf9 insect cells with recombinant baculovirus plasmid

[0117] The recombinant baculovirus plasmids rBac-TBCap, rBac-GP3-GP5-M, and rBac-TBCap-GP3-GP5-M were transfected into monolayer adherent sf9 cells, respectively. The specific transfection methods were as follows:

[0118] (1) Adherent SF9 cells were detached from the culture vessel by pipetting with SF 900III medium and the cell density was adjusted to 1×10⁻⁶. 6 cells / mL;

[0119] (2) Add 2 mL of Grace's Unsupplemented medium per well to 8 × 10⁸ ml of the medium. 5 Add the sf9 cells to a six-well cell culture dish and let stand at room temperature for 30 minutes.

[0120] (3) Dilute 2 μg of purified recombinant baculovirus plasmid with 100 μL of Grace's, Unsupplemented medium into tube A. Mix 100 μL of Grace's, Unsupplemented medium and 8 μL of Cellfectin II Reagent into tube B. Let stand at room temperature for 5 min;

[0121] (4) Mix tube A and tube B thoroughly and incubate at room temperature for 30 minutes;

[0122] (5) Add the mixture from step (4) evenly to the sf9 cells prepared in step (2) and transfect for 4 hours in a 27°C incubator;

[0123] (6) Discard the supernatant, add fresh SF900III culture medium and continue culturing at 27°C until cytopathic effect occurs (approximately 96 hours);

[0124] 2. Isolation of recombinant baculovirus

[0125] After transfection, cell status was observed. When cell abnormalities such as increased cell volume, elongated shape, rough edges, and intracellular vesicles appeared, the cell culture was harvested. The supernatant (P1 generation recombinant baculovirus) was collected by centrifugation at 1000 rpm for 5 min at room temperature. 2% FBS was added to the P1 generation baculovirus, and the culture was stored at -80℃ for later use. The obtained recombinant baculoviruses were named Ac-TBCap, Ac-GP3-GP5-M, and Ac-TBCap-GP3-GP5-M, respectively.

[0126] 3. Passage of recombinant baculoviruses

[0127] To increase the viral titer of recombinant baculovirus, the obtained recombinant baculovirus needs to be passaged 2 to 3 times.

[0128] III. Expression and Identification of Recombinant Proteins

[0129] 1. Indirect immunofluorescence detection of recombinant proteins

[0130] (1) P3 generation recombinant baculovirus Ac-TBCap, Ac-GP3-GP5-M and Ac-TBCap-GP3-GP5-M were used to infect adherent sf9 cells at a dose of MOI=1, with wild-type baculovirus-infected sf9 cells as the control group. The cells were cultured at 27℃ for 72h.

[0131] (2) Remove the cells, discard the culture medium, and wash the cells three times with PBS.

[0132] (3) Add 100 μL of 4% paraformaldehyde fixative and fix at room temperature for 30 min.

[0133] (4) Discard the 4% paraformaldehyde fixative, wash once with PBS, add 100μL of 0.3% Triton X-100 working solution to permeate the cell membrane for 15min.

[0134] (5) Discard 0.3% Triton X-100 working solution, wash 3 times with PBS, add 100 μL of primary antibody, and incubate overnight at 4°C.

[0135] (6) Discard the primary antibody, wash 3 times with PBS, add 100 μL of secondary antibody under light-protected conditions, and incubate at 37°C for 1 h under light-protected conditions.

[0136] (7) Wash with PBS 3 times, 5 min each time, add 100 μL of PBS, and observe under a fluorescence microscope.

[0137] The results showed that specific fluorescence was detected in sf9 cells infected with Ac-TBCap, Ac-GP3-GP5-M, and Ac-GP3-GP5-M-TBCap, while no specific fluorescence was detected in the negative control group. Figure 5 This indicates that the recombinant baculovirus was successfully constructed and can express the recombinant target protein in sf9 cells.

[0138] 2. Expression of recombinant proteins

[0139] High Five cells in the logarithmic growth phase were infected with P3 generation recombinant baculoviruses Ac-TBCap, Ac-GP3-GP5-M, and Ac-GP3-GP5-M-TBCap at an MOI of 1.0 and cultured at 27°C for 72 h. High Five cells without recombinant virus inoculation were used as controls. When most cells showed typical lesions, 80 μL of cell samples were collected, and Western blot was performed to identify recombinant GP3, GP5, M, and TBCcp using a murine His-tagged antibody as the primary antibody.

[0140] The results showed that cell samples infected with Ac-TBCcp exhibited specific bands at 20 kDa and 42 kDa, respectively; cell samples infected with Ac-GP3-GP5-M exhibited specific bands at 28 kDa, 25 kDa, and 20 kDa, respectively; cell samples infected with Ac-GP3-GP5-M-TBCap exhibited specific bands at 28 kDa, 25 kDa, 20 kDa, and 42 kDa, respectively; while High-five cell protein samples infected with wild-type baculovirus did not show any specific bands. Figure 6 This demonstrates that Ac-TBCap, Ac-GP3-GP5-M, and Ac-GP3-GP5-M-TBCap can correctly express the recombinant target protein in High-five cells.

[0141] 3. Optimization of recombinant protein expression conditions

[0142] (1) High-five cells were cultured at a volume of 10 mL / flask until the cell density reached 2 × 10⁻⁶. 6 cells / mL.

[0143] (2) Recombinant P3 generation baculoviruses Ac-TBCap, Ac-GP3-GP5-M, and Ac-GP3-GP5-M-TBCap were seeded into three flasks of High-five cells at MOIs of 2.0, 5.0, and 8.0, respectively, and cultured in a constant temperature shaker at 27°C and 140 rpm. 80 μL of cell samples were collected at 48 h, 72 h, 96 h, and 120 h post-inoculation for Western blot analysis to detect the expression of recombinant proteins GP3, GP5, M, and TBCap.

[0144] The results showed that the optimal expression conditions for recombinant protein TBCap were MOI=5.0, and the protein was harvested 72 hours after inoculation into High-five cells. The optimal expression conditions for recombinant proteins GP3, GP5, and M were MOI=5.0, and the protein was harvested 96 hours after inoculation into High-five cells (partial results are shown in [link to results]). Figure 7 The optimal expression conditions for recombinant proteins GP3, GP5, M, and TBCap were MOI = 5.0, and proteins were harvested 96 hours after inoculation into High-five cells.

[0145] V. Semi-quantitative analysis of recombinant proteins

[0146] The recombinant protein was expressed under the optimal expression conditions determined through the above exploration. Cells were treated at the optimal protein harvest time, collected in 50 mL centrifuge tubes, centrifuged at 5000 rpm for 10 min at 4°C, the supernatant was discarded, and the precipitate was resuspended in 10 mL of PBS. Cells were then thoroughly lysed using sonication, centrifuged at 12000 × g at 4°C for 30 min, and the supernatant was used to prepare protein samples. His-tagged proteins at concentrations of 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, and 50 μg / mL were used as standard proteins. A standard curve was plotted using the standard protein concentrations and the gray values ​​of the bands, and the concentration of the recombinant protein was analyzed semi-quantitatively.

[0147] The results showed that the concentration of recombinant protein PCV2-TBCap after 5-fold concentration of the Ac-TBCap cell culture lysate supernatant was approximately 730 μg / mL, compared to approximately 146 μg / mL in the original cell culture before concentration. The concentrations of PRRSV-GP3, PRRSV-GP5, and PRRSV-M proteins after 5-fold concentration of the Ac-GP3-GP5-M infected cell culture lysate supernatant were approximately 533 μg / mL, 542 μg / mL, and 650 μg / mL, respectively; compared to approximately 107 μg / mL, 108 μg / mL, and 130 μg / mL in the original cell culture before concentration. After 5-fold concentration of the supernatant from the lysate of Ac-TBCap-GP3-GP5-M cell cultures, the protein concentrations of PCV2-TBCAP, PRRSV-GP3, PRRSV-GP5, and PRRSV-M were approximately 525 μg / mL, 250 μg / mL, 293 μg / mL, and 288 μg / mL, respectively; while the concentrations in the original cell culture before concentration were approximately 105 μg / mL, 50 μg / mL, 59 μg / mL, and 58 μg / mL, respectively. Figure 8 ).

[0148] Example 3 Vaccine Preparation

[0149] The recombinant proteins PCV2-TBCap, GP3-GP5-M, and GP3-GP5-M-TBCap obtained in Example 2 were adjusted to 50 μg / mL with PBS (the co-expressed proteins were adjusted according to the concentration of GP3), and emulsified with ISA 201VG adjuvant at a mass ratio of 1:1. The physicochemical properties of the prepared vaccines were tested according to Example 4.

[0150] Example 4: Immune Evaluation Test of Piglets

[0151] Fifteen 4-week-old piglets that tested negative for both PRRSV and PCV2 antigens and antibodies were randomly divided into 5 groups of 3 piglets each. They were immunized via intramuscular injection into the neck. The immunization groupings and dosages are shown in Table 2. The commercially available vaccines used were porcine circovirus type 2 baculovirus vector inactivated vaccine (Boehringer Ingelheim Animal Health, Germany) and porcine reproductive and respiratory syndrome virus inactivated vaccine (Jilin Special Research Biotechnology Co., Ltd.). Two weeks after the initial immunization, a booster immunization was administered at the same dose. Blood samples were collected on days 0, 7, 14, 21, 28, and 35 post-immunization, and serum was separated for the detection of specific antibodies, neutralizing antibodies, and cytokines. Rectal temperature was recorded daily at fixed times after immunization, and clinical symptoms were observed and recorded. Piglets were weighed on days 0 and 28 post-immunization, and the average daily weight gain was calculated.

[0152] Table 2 Grouping of experimental animals

[0153]

[0154] 1. Detection of PRRSV / PCV2 specific antibodies in immunized porcine serum

[0155] Vaccines were prepared by emulsifying ISA201VG adjuvant with the corresponding recombinant proteins PCV2-TBCap, PRRSV-GP3-GP5-M, and PRRSV-GP3-GP5-M+PCV2-TBCap, and the protective effect of the vaccines was evaluated in groups. The specific methods are as follows:

[0156] The levels of PRRSV / PCV2 specific antibodies in the peripheral blood of piglets were measured using the porcine PRRSV IgG indirect ELISA kit (RXYB0002) from Fujian Ruixin Biotechnology Co., Ltd., and the porcine circovirus 2-dCap-ELISA kit (RX-AN500680P) from Wuhan Keqian Biotechnology Co., Ltd., respectively.

[0157] Indirect ELISA was used to detect specific antibodies, with a positive result defined as an OD value greater than 0.4 at a 40-fold serum dilution. Results showed ( Figure 9 Before the first immunization, all piglets were negative for PRRSV-specific antibodies in their serum. After the first immunization, the level of PRRSV-specific antibodies in the immunized piglets gradually increased, becoming positive only after the second immunization, reaching its peak on day 35, with no significant difference between groups (p>0.05). The PRRSV-GP3-GP5-M vaccine group and...

[0158] The PRRSV-GP3-GP5-M+PCV2-TBCap bivalent vaccine group effectively stimulated piglets to produce specific antibodies against PRRSV. Therefore, the PRRSV-GP3-GP5-M protein co-expressed by the recombinant baculovirus system has good immunogenicity and can induce humoral immune responses in the body.

[0159] Specific antibodies were detected using indirect ELISA, with a (SN) / (PN) ratio greater than 0.16 at a 40-fold serum dilution as the positive criterion. Results showed ( Figure 10 Before the first immunization, all piglets were negative for PCV2-specific antibodies in their serum. After the first immunization, the level of PCV2-specific antibodies in the immunized piglets gradually increased, becoming positive only after the second immunization, reaching its peak on day 35, with no significant difference between groups (p>0.05). Both the PCV2-TBCap vaccine group and the PRRSV-GP3-GP5-M+PCV2-TBCap bivalent vaccine immunization group effectively stimulated piglets to produce PCV2-specific antibodies. Therefore, the PCV2-TBCap protein co-expressed by the recombinant baculovirus system has good immunogenicity and can induce humoral immune responses in the body.

[0160] 2. Detection of neutralizing antibodies in immunized pig serum

[0161] One of the most important indicators for evaluating vaccine effectiveness is the titer of neutralizing antibodies in the body. To verify the protective effect induced by recombinant proteins in piglets, a peripheral blood microneutralization assay was performed in vitro to determine the titers of neutralizing antibodies against PRRSV and PCV2 in piglet serum before immunization and on day 35 post-immunization. The specific methods are as follows:

[0162] (1) MARC-145 cells and PK-15 cells in good condition were cultured in 96-well cell culture dishes and grown into monolayers for later use.

[0163] (2) The serum was placed in a 56°C water bath for 30 min to inactivate it and then filtered through a 0.22 μm filter membrane.

[0164] (3) Dilute serum samples from day 0 and day 35 after the first immunization at an initial ratio of 1:10, and then serially dilute the serum to 1:640 at a ratio of 1:2.

[0165] (4) Add 100 TCID to each well 50 The NADC30-Like PRRSV / PCV2 was incubated at 37°C for 2 hours.

[0166] (5) After the mixture from step (4) has been fully reacted into the cell wells for 1 hour, discard the supernatant and add serum-free DMEM medium. Set up 8 replicates for each group.

[0167] (6) Incubate statically for 3-5 days, observe and record the lesioning of cells in each well, and calculate the neutralizing antibody titer (ND) according to the Reed-Muench method. 50 ).

[0168] Depend on Figure 11 It was found that there was no significant difference in the change of anti-PRRSV neutralizing antibodies in piglets before and after immunization in the PBS group (p>0.05). The neutralizing antibody titers in the serum of piglets in the PRRSV-GP3-GP5-M, PCV2-TBCap+PRRSV-GP3-GP5-M, and commercial vaccine groups on day 35 were significantly higher than those before immunization (p<0.0001). The neutralizing antibody titers in the PRRSV-GP3-GP5-M vaccine group and the PCV2-TBCap+PRRSV-GP3-GP5-M vaccine group were lower than those in the commercial vaccine group, which were 0.76 times and 0.73 times lower, respectively. This indicates that PRRSV-GP3-GP5-M and PCV2-TBCap+PRRSV-GP3-GP5-M can induce a certain degree of protection against PRRSV.

[0169] Depend on Figure 11 It was found that there was no significant difference in the change of anti-PCV2 neutralizing antibody in piglets before and after immunization in the PBS group (p>0.05). The neutralizing antibody titers in the serum of piglets in the PCV2-TBCap vaccine group, PCV2-TBCap+PRRSV-GP3-GP5-M vaccine group and commercial vaccine group on day 35 were significantly increased compared with those before immunization (p<0.0001), and there was no significant difference between the groups (p>0.05). This indicates that both PCV2-TBCap vaccine and PCV2-TBCap+PRRSV-GP3-GP5-M vaccine induced the body to produce a protective effect against PCV2.

[0170] 3. Detection of cytokines in immune porcine serum

[0171] To further determine the degree of cellular immunity in piglets after immunization, the concentrations of Th1 cytokines (IFN-γ and IL-2) and Th2 cytokines (IL-4) in the serum of piglets 35 days after the first immunization were detected using ELISA. This was to assess the level of Th1 and Th2 immune responses in piglets after immunization. The specific methods are as follows: The secretion levels of IFN-γ, IL-2, and IL-4 in each group of piglets 28 days after the first immunization were measured using the Jiangsu Enzyme-Immune Pig IFN-γ ELISA Kit (MM-041202), the Pig IL-2 ELISA Kit (MM-042102), and the Pig IL-4 ELISA Kit (MM-041902).

[0172] Depend on Figure 12It can be seen that the serum IL-2 secretion level of piglets in each immunized group was higher than that in the PBS group, and the difference was significant compared with the PBS group (p<0.01), but the difference between the groups was not significant (p>0.05), and the secretion level was comparable.

[0173] Depend on Figure 12 It can be seen that the serum IFN-γ secretion level of piglets in each immunized group was higher than that in the PBS group, and the difference was significant compared with the PBS group (p<0.01), but the difference between the groups was not significant (p>0.05), and the secretion level was comparable.

[0174] Depend on Figure 12 It was found that the IL-4 secretion level of stimulated lymphocytes in piglets in all immunization groups was significantly higher than that in piglets treated with PBS (p<0.001). Among them, there was no significant difference in the IL-4 secretion level induced by the PCV2-TBCap vaccine group and the PRRSV-GP3-GP5-M vaccine group compared with the commercial vaccine group (p>0.05). The IL-4 secretion level in the PCV2-TBCap+PRRSV-GP3-GP5-M group was significantly higher than that in the commercial vaccine immunization group (p<0.01). This indicates that the PCV2-TBCap vaccine group, the PRRSV-GP3-GP5-M vaccine group, and the PCV2-TBCap+PRRSV-GP3-GP5-M vaccine group can all effectively enhance the body's Th2-mediated humoral immune response, with the PCV2-TBCap+PRRSV-GP3-GP5-M group showing better results.

[0175] 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 PRRSV-GP3-GP5-M+PCV2-TBCap recombinant protein, characterized in that consist of recombinant GP5 protein and recombinant GP3 protein, recombinant TBCap protein and recombinant M protein, and the amino acid sequences thereof are shown in SEQ ID No: 1~4 respectively.

2. A nucleic acid encoding the PRRSV-GP3-GP5-M+PCV2-TBCap recombinant protein of claim 1, characterized in that: The nucleotide sequences of the genes of the recombinant GP5 protein, the recombinant GP3 protein, the recombinant TBCap protein and the recombinant M protein are shown in SEQ ID No: 5~8 respectively.

3. A multi-gene expression cassette comprising a gene encoding the PRRSV-GP3-GP5-M+PCV2-TBCap recombinant protein of claim 1 or 2, characterized in that The gene encoding the recombinant GP5 protein and the gene encoding the recombinant GP3 protein are respectively connected with a polyhedrin promoter; the gene encoding the recombinant TBCap protein and the gene encoding the recombinant M protein are connected with a p10 promoter; and the polyhedrin promoter and the p10 promoter are in opposite directions.

4. A recombinant transfer plasmid comprising the multi-gene expression cassette of claim 3, wherein Prepared by the following method: (1) containing Nhe I and Xho The gene sequence encoding the recombinant TBCap protein at the I restriction site and the recombinant transfer plasmid pFBDM-GP5 were respectively used... Nhe I and Xho After digestion with enzyme I and ligation, pFBDM-GP5-TBCap was obtained; the recombinant transfer plasmid pFBDM-GP5 was obtained by ligating the gene encoding the recombinant GP5 protein with pFBDM. (2) the recombinant M protein gene sequence containing Nhe I and Xho I enzyme sites and the recombinant transfer plasmid pFBDM-GP3 are respectively digested by Nhe I and Xho I enzymes and ligated to obtain pFBDM-GP3-M; The group transfer plasmid pFBDM-GP3 is obtained by connecting the gene encoding the recombinant GP3 protein with pFBDM; (3) The expression cassette of the GP5 and TBCap genes is obtained by cutting pFBDM-GP5-TBCap with restriction endonuclease PmeI / AvrII; pFBDM-GP3-M is cut by restriction endonuclease BstZ17I / SpeI; (4) The obtained expression cassette of the GP5 and TBCap genes is connected with the cut pFBDM-GP3-M to obtain the recombinant transfer plasmid pFBDM-GP3-GP5-M-TBCap.

5. A recombinant baculovirus plasmid, characterized in that The DH10Multibac competent cells are obtained by transforming the recombinant transfer plasmid pFBDM-GP3-GP5-M-TBCap of claim 4.

6. A recombinant baculovirus, characterized in that The sf9 insect cells are transfected with the recombinant baculovirus plasmid rBac-TBCap-GP3-GP5-M of claim 5, and the PRRSV-GP3-GP5-M+PCV2-TBCap recombinant protein is obtained by isolation and purification.

7. A method for preparing the PRRSV-GP3-GP5-M+PCV2-TBCap recombinant protein according to claim 1 or 2, characterized in that Comprising the following steps: The High-five cells are transfected with the recombinant baculovirus of claim 6, centrifuged after transfection, and the supernatant is discarded; the cells are resuspended and lysed, centrifuged, and the supernatant is collected to obtain the PRRSV-GP3-GP5-M+PCV2-TBCap recombinant protein.

8. The use of the PRRSV-GP3-GP5-M+PCV2-TBCap recombinant protein of claim 1 or 2 in the preparation of a porcine reproductive and respiratory syndrome and porcine circovirus type 2 bivalent vaccine.

9. A porcine reproductive and respiratory syndrome, porcine circovirus type 2 bivalent vaccine, characterized in that Comprising the PRRSV-GP3-GP5-M+PCV2-TBCap recombinant protein of claim 1 or 2.

10. The vaccine of claim 9, characterized in that: The vaccine further comprises an ISA201VG adjuvant.

Images