A fusion protein containing porcine reproductive and respiratory syndrome virus protein, subunit vaccine and construction method and application

Abstract

This invention belongs to the field of bioengineering technology. It provides a fusion protein containing porcine reproductive and respiratory syndrome virus (PRRSV) protein, a subunit vaccine, its construction method, and its applications. This invention constructs a fusion protein and its encoding gene by linking an Igκ signal peptide to the N-terminus of the PRRSV protein, and establishes a recombinant vector and recombinant cell line. The PRRSV fusion protein encoding gene, recombinant vector, and recombinant cell line constructed by this invention can efficiently amplify and express exogenous genes, making the expressed PRRSV protein easy to isolate and purify, and capable of forming a stable dimer morphology. Using the expressed PRRSV fusion protein as an antigen to prepare a subunit vaccine, inoculation into mice elicits a strong specific humoral immune response and effectively increases the level of neutralizing antibodies in the animals.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and gene technology, and relates to a fusion protein containing porcine reproductive and respiratory syndrome virus (PRRSV) protein, a subunit vaccine, its construction method and application, specifically involving a fusion protein including PRRSV Gp4Fc protein, Gp5Fc protein or MFc protein and its related encoding gene, recombinant vector, recombinant cell line and its construction method and subunit vaccine. Background Technology

[0002] The pathogen of porcine reproductive and respiratory syndrome (PRRS) is porcine reproductive and respiratory syndrome virus (PRRSV). This virus is highly contagious and can cause symptoms such as respiratory distress in pigs, piglet mortality, reproductive disorders and abortion in sows. Infection with the virus can lead to immunosuppression and various complications, causing huge economic losses to the pig industry.

[0003] The genome of PRRSV is approximately 15.4 kb in length, encoding 16 non-structural proteins (nsp1α, nsp1β, nsp2, nsp2TF, nsp2N, nsp3-6, nsp7α, nsp7β, and nsp8-12) and 8 structural proteins (GP2a / ORF2a, GP3 / ORF3, GP4 / ORF4, GP5 / ORF5, E / ORF2b, 5a / ORF5a, M / ORF6, and N / ORF7). The PRRSV GP5 protein is a glycosylated membrane protein that induces the production of neutralizing antibodies. A non-neutralizing epitope (Epitope A, 27-30 aa) precedes the neutralizing epitope (Epitope B, 37-35 aa). Animals produce antibodies against the A epitope first, followed by antibodies against the B epitope, thus delaying the neutralizing effect of the B epitope.

[0004] Currently, commercial vaccines against PRRS are mainly modified live vaccines (MLV) and inactivated vaccines. MLV vaccines against PRRSV primarily protect against homologous strains, but offer poor protection against heterologous strains and are prone to safety issues such as virulence reversion, recombinant infections, and high variability. Compared to MLV vaccines, inactivated PRRSV vaccines are widely recognized for their safety, but they suffer from drawbacks such as poor efficacy against heterologous strains, higher dosage requirements, lower protective efficacy, more immunization cycles, and higher costs. In addition to the already marketed MLV and inactivated vaccines, there are also DNA vaccines, subunit vaccines, or viral vector vaccines containing other viruses. However, these vaccines are generally less effective than MLV and do not provide complete protection. Therefore, developing a safe and effective PRRSV vaccine is crucial for the prevention and control of PRRS. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to provide a fusion protein containing porcine reproductive and respiratory syndrome virus (PRRSV) protein, a subunit vaccine, and its construction method and application, thereby providing an effective and safe immunogen for the development of PRRSV vaccines, enriching the types of immunogens, and thus achieving safe and effective vaccine development to better improve the prevention and control of PRRS.

[0006] This invention provides a fusion protein containing porcine reproductive and respiratory syndrome virus (PRRSV) protein, the fusion protein comprising the PRSV protein and an Igκ signal peptide linked to the N-terminus of the PRSV protein;

[0007] The amino acid sequence of the Igκ signal peptide is shown in SEQ ID NO:20;

[0008] The porcine reproductive and respiratory syndrome virus (PRRSV) proteins include Gp4Fc, Gp5Fc, or MFc.

[0009] Preferably, the fusion protein includes one or more of Gp4Fc fusion protein, Gp4Fc fusion protein and MFc fusion protein;

[0010] The Gp4Fc fusion protein has the amino acid sequence shown in SEQ ID NO:6, or a protein obtained by substituting and / or deleting and / or adding amino acid residues of SEQ ID NO:6 that has more than 80% identity with the protein shown in SEQ ID NO:6 and has the same function; the Gp5Fc fusion protein has the amino acid sequence shown in SEQ ID NO:7, or a protein obtained by substituting and / or deleting and / or adding amino acid residues of SEQ ID NO:7 that has more than 80% identity with the protein shown in SEQ ID NO:7 and has the same function; the MFc fusion protein has the amino acid sequence shown in SEQ ID NO:8, or a protein obtained by substituting and / or deleting and / or adding amino acid residues of SEQ ID NO:8 that has more than 80% identity with the protein shown in SEQ ID NO:8 and has the same function.

[0011] The present invention provides a gene encoding the above-mentioned fusion protein, the gene including a gene encoding a porcine reproductive and respiratory syndrome virus protein and a gene encoding an Igκ signal peptide linked to the 5' end of the gene encoding the porcine reproductive and respiratory syndrome virus protein.

[0012] The nucleotide sequence of the gene encoding the Igκ signal peptide is shown in SEQ ID NO:2.

[0013] Preferably, the coding gene further includes an upstream restriction enzyme recognition sequence linked to the 5' end of the Igκ signal peptide coding gene, a downstream restriction enzyme recognition sequence linked to the 3' end of the coding gene for the porcine reproductive and respiratory syndrome virus (PRRSV) protein, and a coding gene for a 6×His tag; the coding gene for the PRSV protein includes a coding gene for Gp4Fc, a coding gene for Gp5Fc, or a coding gene for MFc; the nucleotide sequence of the coding gene for Gp4Fc is shown in SEQ ID NO:21; the nucleotide sequence of the coding gene for Gp5Fc is shown in SEQ ID NO:22; and the nucleotide sequence of the coding gene for MFc is shown in SEQ ID NO:23.

[0014] Preferably, the nucleotide sequence encoding the gene includes one or more of the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:5.

[0015] The present invention provides a recombinant vector, wherein the coding gene of any one of the above-mentioned technical solutions is inserted into the recombinant vector.

[0016] The present invention provides a recombinant cell line comprising the above-mentioned recombinant vector.

[0017] This invention provides a method for constructing a recombinant cell line, comprising the following steps:

[0018] The recombinant vector and helper plasmid were packaged into lentiviruses, and the packaged lentiviruses were transferred into host cells to obtain recombinant cell lines.

[0019] The present invention also provides the application of the recombinant cell lines constructed by the above-mentioned fusion protein, encoding gene, recombinant vector or recombinant cell line or method for constructing recombinant cell lines in the preparation of vaccines for the prevention of porcine reproductive and respiratory syndrome.

[0020] The present invention also provides a porcine reproductive and respiratory syndrome virus subunit vaccine, comprising an antigen and an adjuvant, wherein the antigen comprises the aforementioned fusion protein.

[0021] Beneficial effects of the invention:

[0022] This invention relates to a fusion protein containing porcine reproductive and respiratory syndrome virus (PRRSV) protein, obtained by linking an Igκ signal peptide to the N-terminus of the protein's amino acid sequence. The fusion protein of this invention can form a stable dimer morphology. Using this fusion protein as an antigen protein in the preparation of a subunit vaccine, the resulting subunit vaccine, after inoculation into mice, can elicit a high level of specific humoral immune response against PRSV, while also effectively increasing the level of neutralizing antibodies and effectively blocking the infection route of PRSV.

[0023] The present invention also provides the encoding gene of the fusion protein, a recombinant vector, and a recombinant cell line. The recombinant cell line provided by the present invention, as a recombinant expression system, can efficiently amplify and express exogenous genes, making the expressed protein easy to isolate and purify, and the expressed porcine reproductive and respiratory syndrome virus protein can form a stable dimer morphology.

[0024] The present invention also provides a subunit vaccine for porcine reproductive and respiratory syndrome virus (PRRSV). This subunit vaccine enriches the types of immunogens for PRSV vaccines and can serve as a promising candidate subunit vaccine for the prevention of PRSV, with high clinical application value and good research prospects. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1Schematic diagram of the structure of the PRRSVGp4Fc, PRRSVGp5Fc and PRRSVMFc fusion protein;

[0027] Figure 2 The spectrum of the pTRIP-MCS-IRES-GS vector;

[0028] Figure 3 The images show the PCR identification of the plasmids pTRIP-MCS-IRES-GS-PRRSV-Gp4Fc, pTRIP-MCS-IRES-GS-PRRSV-Gp5Fc, and pTRIP-MCS-IRES-GS-PRRSV-MFc. Figure 3 In the diagram, A represents the PCR identification product of the transfer vector pTRIP-MCS-IRES-GS-PRRSV-Gp4Fc; M: DL2000 DNA Marker; 1-6: Gp4Fc PCR identification products. Figure 3 In the diagram, B represents the PCR identification product of the transfer vector pTRIP-MCS-IRES-GS-PRRSV-Gp5Fc; M: DL2000 DNA Marker; 1-6: Gp5Fc PCR identification products. Figure 3 In the diagram, C represents the PCR identification product of the transfer vector pTRIP-MCS-IRES-GS-PRRSV-MFcPCR; M: DL2000 DNA Marker, 1-6: MFc PCR identification products;

[0029] Figure 4 Figure 1. Western blotting results of 293T transfection with the target plasmids pTRIP-MCS-IRES-GS-PRRSV-Gp4Fc, pTRIP-MCS-IRES-GS-PRRSV-Gp5Fc and pTRIP-MCS-IRES-GS-PRRSV-MFc.

[0030] Figure 5 Western blotting results of transduced Lenti-PRRSV-Gp4Fc, Lenti-PRRSV-Gp5Fc, or Lenti-PRRSV-MFc after CHO-GS-KO; Marker: Protein molecular weight standard;

[0031] Figure 6 The results of drug treatment screening for CHO-GS-KO-PRRSV-Gp4Fc, CHO-GS-KO-PRRSV-Gp5Fc, and CHO-GS-KO-PRRSV-MFc cell lines; among them... Figure 6In the diagram, A represents the screening results of drug administration for the CHO-GS-KO-PRRSV-Gp4Fc cell line; M represents the protein molecular weight standard; 1–12 represent samples from different wells in a 12-well plate. Figure 6 In the diagram, B represents the screening results of drug administration for the CHO-GS-KO-PRRSV-Gp5Fc cell line; M represents the protein molecular weight standard; 1–12 represent samples from different wells in a 12-well plate. Figure 6 In this context, C represents the drug screening results of the CHO-GS-KO-PRRSV-MFc cell line; M represents the protein molecular weight standard; and 1–12 represent samples from different wells in a 12-well plate.

[0032] Figure 7 The results of immunofluorescence assays for CHO-GS-KO-PRRSV-Gp4Fc, CHO-GS-KO-PRRSV-Gp5Fc, and CHO-GS-KO-PRRSV-MFc cell lines are shown in the figure.

[0033] Figure 8 The figure shows the purification results of CHO-GS-KO-PRRSV-Gp4Fc, CHO-GS-KO-PRRSV-Gp5Fc, and CHO-GS-KO-PRRSV-MFc proteins. Figure 8 In the image, A represents the SDS-PAGE identification result of the purified PRRSV-Gp4Fc protein; Marker: protein molecular weight standard; Figure 8 In the image, B represents the SDS-PAGE identification result of the purified PRRSV-Gp5Fc protein; Marker: protein molecular weight standard; Figure 8 In the image, C represents the SDS-PAGE identification result of the purified PRRSV-MFc protein; Marker: protein molecular weight standard;

[0034] Figure 9 Characterization diagram of PRRSV-Gp4Fc, PRRSV-Gp5Fc and PRRSV-MFc fusion protein dimers M: protein molecular weight standard;

[0035] Figure 10 The results show the detection of PRRSV-specific antibody levels in mice at different time points after immunization; among them, Figure 10 In the figure, A represents the detection results of PRRSV-Gp4Fc specific antibody levels; Figure 10 B in the figure represents the result of PRRSV-Gp5Fc specific antibody level detection; Figure 10 In this context, C represents the result of PRRSV-MFc specific antibody level detection;

[0036] Figure 11 The graph shows the results of humoral immunity against the PRRSV-Q strain at different time points after mouse immunization.

[0037] Figure 12 This is a graph showing the results of PRRSV neutralizing antibody level detection. Detailed Implementation

[0038] This invention provides a fusion protein containing porcine reproductive and respiratory syndrome virus (PRRSV) protein, the fusion protein comprising the PRSV protein and an Igκ signal peptide linked to the N-terminus of the PRSV protein;

[0039] The amino acid sequence of the Igκ signal peptide is shown in SEQ ID NO:20, and the specific amino acid sequence is as follows:

[0040] N-METDTLLLWVLLLWVPGSTG-C;

[0041] The porcine reproductive and respiratory syndrome virus (PRRSV) proteins include Gp4Fc, Gp5Fc, or MFc.

[0042] In this invention, the fusion protein preferably includes one or more of Gp4Fc fusion protein, Gp5Fc fusion protein and MFc fusion protein; the amino acid sequence of the Gp4Fc fusion protein is preferably as shown in SEQ ID NO:6; the amino acid sequence of the Gp5Fc fusion protein is preferably as shown in SEQ ID NO:7; and the amino acid sequence of the MFc fusion protein is preferably as shown in SEQ ID NO:8.

[0043] In this invention, the specific amino acid sequence of SEQ ID NO:6 is as follows:

[0044]

[0045] In this invention, the amino acid sequence of the Gp4Fc fusion protein may also be a protein with more than 80% identity and the same function as the protein shown in SEQ ID NO:6, obtained by substituting and / or deleting and / or adding the amino acid residues of SEQ ID NO:6.

[0046] The specific amino acid sequence of SEQ ID NO:7 is shown below:

[0047]

[0048] In this invention, the amino acid sequence of the Gp5Fc fusion protein may also be a protein with more than 80% identity and the same function as the protein shown in SEQ ID NO:7, obtained by substituting and / or deleting and / or adding the amino acid residues of SEQ ID NO:7.

[0049] The specific amino acid sequence of SEQ ID NO:8 is shown below:

[0050]

[0051] In this invention, the amino acid sequence of the MFc fusion protein may also be a protein with more than 80% identity and the same function as the protein shown in SEQ ID NO:8, obtained by substituting and / or deleting and / or adding the amino acid residues of SEQ ID NO:8.

[0052] The present invention provides the coding gene of the fusion protein described in the above technical solution, wherein the coding gene includes the coding gene of porcine reproductive and respiratory syndrome virus protein and the coding gene of Igκ signal peptide linked to the 5' end of the coding gene of porcine reproductive and respiratory syndrome virus protein;

[0053] The nucleotide sequence of the gene encoding the Igκ signal peptide is shown in SEQ ID NO:2, and the specific nucleotide sequence is as follows:

[0054] 5'-atggaaacagatacactcctcctctgggtgctgctcctctgggtgccaggatctacagga-3'.

[0055] Furthermore, the coding gene described in the above technical solution preferably also includes an upstream restriction enzyme recognition sequence linked to the 5' end of the Igκ signal peptide coding gene, a downstream restriction enzyme recognition sequence linked to the 3' end of the coding gene for the porcine reproductive and respiratory syndrome virus protein, and a coding gene for a 6×His tag;

[0056] The coding genes for the porcine reproductive and respiratory syndrome virus protein include the coding gene for Gp4Fc, the coding gene for Gp5Fc, or the coding gene for MFc.

[0057] The nucleotide sequence of the gene encoding Gp4Fc is shown in SEQ ID NO:21;

[0058] The nucleotide sequence of the gene encoding Gp5Fc is shown in SEQ ID NO:22;

[0059] The nucleotide sequence of the gene encoding the MFc is shown in SEQ ID NO:23.

[0060] The specific nucleotide sequence of SEQ ID NO:21 is shown below:

[0061]

[0062] The specific nucleotide sequence of SEQ ID NO:22 is shown below:

[0063]

[0064] The specific nucleotide sequence of SEQ ID NO:23 is shown below:

[0065]

[0066] In this invention, the nucleotide sequence of the 6×His tag is preferably as shown in SEQ ID NO:3;

[0067] The specific nucleotide sequence of SEQ ID NO:3 is shown below:

[0068] 5'-catcaccatcaccatcac-3'.

[0069] In this invention, the upstream enzyme digestion recognition sequence is preferably BamHI; the downstream enzyme digestion recognition sequence is preferably Mlu I.

[0070] In this invention, the BamHI recognition sequence is preferably as follows:

[0071] 5'-GGATCC-3'.

[0072] In this invention, the recognition sequence of Mlu I is preferably as follows:

[0073] 5'-ACGCGT-3'.

[0074] Furthermore, the nucleotide sequence encoding the gene described in the above technical solution preferably includes one or more of the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:5.

[0075] The specific nucleotide sequence of SEQ ID NO:1 is shown below:

[0076]

[0077] The specific nucleotide sequence of SEQ ID NO:4 is shown below:

[0078]

[0079] The specific nucleotide sequence of SEQ ID NO:5 is shown below:

[0080]

[0081] The present invention provides a recombinant vector, wherein the recombinant vector is inserted with the coding gene described in any one of the above technical solutions.

[0082] In this invention, the original vector of the recombinant vector preferably includes a eukaryotic transfer vector, more preferably pTRIP-MCS-IRES-GS; the coding gene is preferably inserted between the BamHI and Mlu I restriction sites.

[0083] In this invention, the preferred method for preparing the recombinant vector includes the following steps: cloning the coding genes of the porcine reproductive and respiratory syndrome virus (PRRSV) proteins described in the above technical solution into eukaryotic transfer vectors, performing enzyme digestion and ligation, transforming the ligation products into competent cells, extracting plasmids, and obtaining the recombinant vector. This invention does not impose any particular limitation on the cloning method; any cloning method well-known in the art can be used.

[0084] The present invention provides a recombinant cell line comprising the recombinant vector described in the above technical solution.

[0085] In this invention, the original cells of the recombinant cell line preferably include eukaryotic cells, animal cells, or prokaryotic cells; the eukaryotic cells more preferably include yeast cells or Aspergillus; the animal cells more preferably include mammalian cells or insect cells; and the prokaryotic cells more preferably include Escherichia coli or Bacillus subtilis. In a specific embodiment of this invention, the cells preferably include CHO-GS-KO cells, and more preferably include CHO-GS-KO cells suspended in serum-free medium containing L-Glutamine.

[0086] The present invention also provides a method for constructing the above-mentioned recombinant cell line, comprising the following steps:

[0087] The recombinant vector and helper plasmid described in the above technical solution are packaged into lentivirus, and the packaged lentivirus is transferred into host cells to obtain a recombinant cell line.

[0088] In this invention, the auxiliary plasmid preferably includes PSPA×2 and PMD2G.

[0089] In this invention, the method for packaging lentiviruses preferably includes the following steps: co-transfecting the recombinant vector and helper plasmids PSPA×2 and PMD2G into 293T cells, culturing them, and collecting the recombinant lentiviruses Lenti-PRRSV-Gp4Fc, Lenti-PRRSV-Gp5Fc and Lenti-PRRSV-MFc expressing PRRSV-Gp4Fc, PRRSV-Gp5Fc and PRRSV-MFc proteins, respectively.

[0090] In this invention, the lentivirus packaging transfection system preferably includes a transduction reagent and a mixed plasmid; the mass ratio of the transduction reagent to the mixed plasmid is further preferably 2:1.

[0091] In this invention, the preferred mass concentration ratio of the recombinant vector to the helper plasmid is: recombinant vector: PSPA×2: PMD2G = 1:0.65:0.35.

[0092] In this invention, the method for transducing the packaged lentivirus into host cells preferably includes the following steps: co-culturing the packaged lentivirus and host cells under the action of a transduction reagent. This invention does not impose any particular limitation on the transduction reagent; any transduction reagent well-known in the art can be used. In a specific embodiment of this invention, the transduction reagent preferably includes a polybrene reagent.

[0093] In this invention, the original expression vector after packaging is transferred into host cells preferably includes a methionine sulfoxide (MSX) pressure screening step.

[0094] This invention provides an application of the recombinant cell line constructed by the fusion protein, encoding gene, recombinant vector, recombinant cell line or the method for constructing the recombinant cell line described in the above technical solution in the preparation of a vaccine for the prevention of porcine reproductive and respiratory syndrome.

[0095] The present invention provides a porcine reproductive and respiratory syndrome virus subunit vaccine, comprising an antigen and an adjuvant, characterized in that the antigen comprises one or more of the fusion proteins described in the above-described technical solution.

[0096] In this invention, the volume ratio of the immunoantigen to the adjuvant is preferably 1:1. When the immunoantigen is a monoantigen, the final concentration is preferably 30-50 μg / mL, more preferably 40 μg / mL; when the immunoantigen is divalent, the final concentration is preferably 60-100 μg / mL, more preferably 80 μg / mL; when the immunoantigen is trivalent, the final concentration is preferably 90-150 μg / mL, more preferably 120 μg / mL. In this invention, when the immunoantigen is divalent, the two immunoantigens are preferably mixed in a volume ratio of 1:1; when the immunoantigen is trivalent, the three immunoantigens are preferably mixed in a volume ratio of 1:1:1. This invention does not have any special limitations on the type of adjuvant; any adjuvant well known in the art can be used. In a specific embodiment of this invention, the adjuvant preferably includes Montanide ISA. TM 201. Adjuvant. In this invention, the method of administering the subunit vaccine preferably includes two immunizations, with each immunization dose preferably being 100-300 μL, more preferably 200 μL, and the two immunizations preferably being 3 weeks apart.

[0097] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a fusion protein containing porcine reproductive and respiratory syndrome virus protein, a subunit vaccine, its construction method, and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0098] Example 1

[0099] The construction methods of the transfer vectors pTRIP-MCS-IRES-GS-PRRSV-Gp4Fc, pTRIP-MCS-IRES-GS-PRRSV-Gp5Fc, and pTRIP-MCS-IRES-GS-PRRSV-MFc expressing porcine reproductive and respiratory syndrome virus (PRRSV) Gp4Fc, PRRSV Gp5Fc, and PRRSV MFc are illustrated in the diagram below. Figure 1 .

[0100] 1. Construction method of transfer vector pTRIP-MCS-IRES-GS-PRRSV-Gp4Fc expressing porcine reproductive and respiratory syndrome virus (PRRSV) Gp4Fc fusion protein

[0101] (1) Synthesis of PRRSVGp4Fc fusion gene sequence

[0102] PRRSV Gp4 protein was artificially synthesized and its codons were optimized. The signal peptide Igκ (SEQ ID NO:2) was added to the front end of the synthesized PRRSV Gp4. The nucleotide sequences of PRRSV Gp4 and IgG3Fc (SEQ ID NO:9, 5'-gcagaggtcctgggagcaccgtcggtcttcctcttccctccaaaacccaaggacatcctcatgatctcccggacacccaaggtcacgtgcgtggtgg-3') were linked together using Linker (SEQ ID NO:10, 5'-ggatcaggcgggggtgggtccggaggaggtggctcgggatct-3') and Hinge (SEQ ID NO:11, 5'-gacatcgaaccccccacacccatctgtcccgaaatttgctcatgcccagct-3') to obtain the target sequence PRRSVp4-IgG3Fc.

[0103] Primers were designed to add the restriction enzyme site BamHI to the 5' end of the PRRSV Gp4-IgG3Fc nucleotide sequence and the restriction enzyme sites MluI and 6×His (SEQ ID NO:3) to the 3' end. The specific primer sequences are shown in Table 1.

[0104] Table 1 Primer sequences used for BamHI-Igκ-PRRSV Gp4Fc-MluI gene amplification

[0105] Primer name Primer sequence Gp4-F 5'-actggatccgccgccaccatggaaacagatacactcctcctc-3'(SEQ ID NO:12) Fc-R 5'-tatacgcgttcactagtgatggtgatggtgatgagagcccga-3' (SEQ ID NO: 13)

[0106] The PCR amplification system and reaction procedure for the recombinant fragment BamHI-Igκ-PRRSV Gp4Fc-MluI are shown in Tables 2 and 3.

[0107] Table 2 PCR amplification system

[0108] reagents volume PrimerStarmix 25μL Gp4-F 2μL Fc-R 2μL Template DNA 5μL Sterilized ultrapure water 16μL Total volume 50μL

[0109] Table 3 PCR amplification reaction procedure

[0110]

[0111] The sequence BamHI-Igκ-PRRSV Gp4Fc-MluI (SEQ ID NO:1) was obtained by PCR amplification.

[0112] (2) Enzyme digestion and ligation assay

[0113] The BamHI-Igκ-PRRSV Gp4Fc-MluI obtained in step (1) and the eukaryotic transfer vector pTRIP-MCS

[0114] -IRES-GS was double-digested with BamHI and Mlu I restriction endonucleases under the following conditions: 37℃ for 3 hours. The digestion system is shown in Table 4.

[0115] Table 4 Double enzyme digestion system

[0116]

[0117] The DNA gel recovery kit from Tiangen Biotech (Beijing) Co., Ltd. was used to recover the enzyme digestion products, which were then subjected to a ligation reaction. The ligation reaction conditions were: 16℃ for 6 hours, and the ligation reaction system is shown in Table 5.

[0118] Table 5 Connection Reaction System

[0119]

[0120] The product of the above ligation reaction was transformed into DH5α competent cells (purchased from Sangon Biotech). The cells were cultured on LB solid medium containing ampicillin (purchased from Sangon Biotech) for 12 h. Positive colonies were selected and inoculated into LB liquid medium containing ampicillin. After culturing at 37°C and 180 r / min for 12–14 h, plasmids were extracted and then identified by PCR.

[0121] The result is as follows Figure 3 As shown in A, the plasmid was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The recombinant plasmid with correct sequencing was named: pTRIP-MCS-IRES-GS-PRRSV Gp4Fc.

[0122] 2. Construction method of transfer vector pTRIP-MCS-IRES-GS-PRRSV-Gp5Fc expressing porcine reproductive and respiratory syndrome virus (PRRSV) Gp5Fc fusion protein

[0123] (1) Synthesis of PRRSVGp5 gene sequence

[0124] The nucleotide sequence of porcine reproductive and respiratory syndrome virus (PRRSVGp5) was artificially synthesized and codons were optimized. Primers were designed to add the restriction enzyme site BamHI to the 5' end of the PRRSVGp5 nucleotide sequence and to add the Ig3Fc sequence (SEQ ID NO:9), the restriction enzyme site MluI, and 6×His (SEQ ID NO:3) to the 3' end. The nucleotide sequences of PRRSVGp5 and IgG3Fc were connected by a linker (SEQ ID NO:10) and a hinge (SEQ ID NO:11). The specific primer sequences are shown in Table 6.

[0125] Table 6 Primer sequences for PRRSV Gp5Fc gene amplification

[0126] Primer name Primer sequence Gp5-F 5'-actggatccgccgccaccatggaaacagatacactcctcctc-3'(SEQ ID NO:14) Gp5-Fc-R 5'-ccacccccgcctgatcccaggcggccccacagtt-3'(SEQ ID NO:15) Gp5-Fc-F 5'-aactgtggggccgcctgggatcaggcgggggtgg-3'(SEQ ID NO:16) Fc-R 5'-tatacgcgttcactagtgatggtgatggtgatgagagcccga-3' (SEQ ID NO: 13)

[0127] The PCR amplification system and reaction procedure for the recombinant fragment BamHI-Igκ-PRRSV Gp5-MluI are shown in Tables 7 to 10.

[0128] Table 7 PCR amplification system 1

[0129] reagents volume PrimerStarmix 25μL Gp5-F 2μL Gp5-Fc-R 2μL Template DNA 5μL Sterilized ultrapure water 16μL Total volume 50μL

[0130] Table 8 PCR amplification system 2

[0131] reagents volume PrimerStarmix 25μL Gp5-Fc-F 2μL Fc-R 2μL Template DNA 5μL Sterilized ultrapure water 16μL Total volume 50μL

[0132] Table 9 PCR amplification system 3

[0133] reagents volume PrimerStarmix 25μL Gp5-F 2μL Fc-R 2μL Template DNA 5μL Sterilized ultrapure water 16μL Total volume 50μL

[0134] Table 10 PCR Amplification Reaction Procedure

[0135]

[0136] The sequence BamHI-Igκ-PRRSVGp5Fc-MluI (SEQ ID NO:4) was obtained by PCR amplification.

[0137] (2) Enzyme digestion and ligation assay

[0138] The BamHI-Igκ-PRRSV Gp5Fc-MluI obtained in step (1) and the eukaryotic transfer vector pTRIP-MCS

[0139] -IRES-GS was double-digested with BamHI and Mlu I restriction endonucleases under the following conditions: 37℃ for 3 hours. The digestion system is shown in Table 11.

[0140] Table 11 Double Enzyme Digestion System

[0141]

[0142] The DNA gel recovery kit from Tiangen Biotech (Beijing) Co., Ltd. was used to recover the enzyme digestion products, which were then subjected to a ligation reaction. The ligation reaction conditions were: 16℃ for 6 hours, and the ligation reaction system is shown in Table 12.

[0143] Table 12 Connection Reaction System

[0144]

[0145] The product of the above ligation reaction was transformed into DH5α competent cells (purchased from Sangon Biotech). The cells were cultured on LB solid medium containing ampicillin (purchased from Sangon Biotech) for 12 h. Positive colonies were selected and inoculated into LB liquid medium containing ampicillin. After culturing at 37°C and 180 r / min for 12–14 h, plasmids were extracted and then identified by PCR.

[0146] The result is as follows Figure 3 As shown in B, the plasmid was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The recombinant plasmid with correct sequencing was named: pTRIP-MCS-IRES-GS-PRRSV Gp5Fc.

[0147] 3. Construction method of transfer vector pTRIP-MCS-IRES-GS-PRRSV-MFc expressing porcine reproductive and respiratory syndrome virus (PRRSV) MFc fusion protein

[0148] (1) Synthesis of PRRSVM gene sequence

[0149] The nucleotide sequence of porcine reproductive and respiratory syndrome virus (PRRSVM) was artificially synthesized and codon optimized. Primers were designed to add the restriction enzyme site BamHI to the 5' end of the PRRSVM nucleotide sequence and the Ig3Fc sequence (SEQ ID NO: 9) to the 3' end.

[0150] The nucleotide sequences of PRRSV Gp4 and IgG3Fc are linked by a linker (SEQ ID NO:10) and a hinge (SEQ ID NO:11). The specific primer sequences are shown in Table 13.

[0151] Table 13 Primer sequences for PRRSV MFc gene amplification

[0152]

[0153] The PCR amplification system and reaction procedure for the recombinant fragment BamHI-Igκ-PRRSVM-MluI are shown in Tables 14 to 17.

[0154] Table 14 PCR amplification system 1

[0155] reagents volume PrimerStarmix 25μL MF 2μL M-Fc-R 2μL Template DNA 5μL Sterilized ultrapure water 16μL Total volume 50μL

[0156] Table 15 PCR amplification system 2

[0157] reagents volume PrimerStarmix 25μL M-Fc-F 2μL Fc-R 2μL Template DNA 5μL Sterilized ultrapure water 16μL Total volume 50μL

[0158] Table 16 PCR Amplification System 3

[0159] reagents volume PrimerStarmix 25μL MF 2μL Fc-R 2μL Template DNA 5μL Sterilized ultrapure water 16μL Total volume 50μL

[0160] Table 17 PCR Amplification Reaction Procedure

[0161]

[0162] The sequence BamHI-Igκ-PRRSVMFc-MluI (SEQ ID NO:5) was obtained by PCR amplification.

[0163] (2) Enzyme digestion and ligation assay

[0164] The BamHI-Igκ-PRRSVMFc-MluI obtained in step (1) and the eukaryotic transfer vector pTRIP-MCS-IRES-GS were double-digested with BamHI and MluI restriction endonucleases. The digestion conditions were: 37℃ for 3h. The digestion system is shown in Table 18.

[0165] Table 18 Double Enzyme Digestion System

[0166]

[0167] (The eukaryotic transfer vector pTRIP-MCS-IRES-GS was preserved in the laboratory.)

[0168] The DNA gel recovery kit from Tiangen Biotech (Beijing) Co., Ltd. was used to recover the enzyme digestion products, which were then subjected to a ligation reaction. The ligation reaction conditions were: 16℃ for 6 hours, and the ligation reaction system is shown in Table 19.

[0169] Table 19 Connection Reaction System

[0170] reagents volume BamHI-Igκ-PRRSVMFc-MluI enzyme digestion product 6μL pTRIP-MCS-IRES-GS enzyme digestion products 1μL 10×T4 DNA ligase Buffer 2μL T4 DNA ligase 1μL Sterilized ultrapure water 10μL Total volume 20μL

[0171] The product of the above ligation reaction was transformed into DH5α competent cells (purchased from Sangon Biotech). The cells were cultured on LB solid medium containing ampicillin (purchased from Sangon Biotech) for 12 h. Positive colonies were selected and inoculated into LB liquid medium containing ampicillin. After culturing at 37°C and 180 r / min for 12–14 h, plasmids were extracted and then identified by PCR.

[0172] The result is as follows Figure 3 As shown in C, the plasmid was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The recombinant plasmid with correct sequencing was named: pTRIP-MCS-IRES-GS-PRRSVMFc.

[0173] Example 2 Packaging of Lenti-PRRSV Gp4Fc, Lenti-PRRSV Gp5Fc and Lenti-PRRSV MFc

[0174] 293T cells were cultured in a cell culture incubator at 37°C and 5% CO2 using DMEM (Cytiva) medium containing 10% newborn fetal bovine serum (purchased from CB) and 1% penicillin-dextrose antibody. When the cell adhesion concentration reached approximately 70%, the target plasmid pTRIP-MCS-IRES-GS-PRRSV Gp4Fc, pTRIP-MCS-IRES-GS-PRRSV Gp5Fc, or pTRIP-MCS-IRES-GS-PRRSV MFc was co-transfected into 293T cells with helper plasmids PSPA×2 and PMD2G (target plasmid: PSPA×2: PMD2G = 1:0.65:0.35). Negative control wells were also included. After 48 hours of culture, the supernatant from the transfected wells and the negative control wells, along with the 293T cells, were collected for Western blotting identification.

[0175] The results are as follows Figure 4As shown, the expression of Gp4Fc, Gp5Fc, or MFc proteins was detected in both the culture supernatant and 293T cells. The resulting lentiviruses were named Lenti-PRRSV Gp4Fc, Lenti-PRRSV Gp5Fc, and Lenti-PRRSVMFc.

[0176] Example 3: Construction of CHO-GS-KO cell line expressing porcine reproductive and respiratory syndrome virus (PRRSV) Gp4Fc, PRRSV Gp5Fc, or PRRSV MFc fusion protein

[0177] CHO-GS-KO cells were cultured in suspension in VegaCHO™ Medium (OPM Biosciences) containing 6 mM L-Glutamine at 37°C, 5% CO2, and 140 rpm on a shaker. Transduction assays were performed when the cell density reached 2 × 10⁶ cells / mL. 1 mL of cells was seeded into each well of a 6-well plate, and 1.4 μL of Polybrene (Yeasen) transduction reagent was added to each well. Then, 700 μL of Lenti-PRRSV Gp4Fc, Lenti-PRRSV P5Fc, or Lenti-PRRSV MFc was added to each well. After 48 h, 40 μL of the culture supernatant and cells were collected for Western blotting.

[0178] The results are as follows Figure 5 As shown, PRRSVGp4Fc or PRRSVFc proteins were detected in the supernatant, and PRRSV Gp5Fc protein expression was detected in the cell pellet.

[0179] After confirming the expression of the target protein by Western blotting, recombinant cell lines were screened. Cells that had been correctly transduced were seeded into 6-well plates at a density of 4 × 10⁶ cells / well. 4 cells / mL. Simultaneously, different concentrations (0 μM, 25 μM, and 50 μM) of methionine sulfoxide (MSX) were added to the culture medium for screening positive cells. After 72 h, 40 μL of supernatant and cell samples were collected for Western blotting. Screening was continued under pressure for 6 passages, and the results are as follows: Figure 6 As shown.

[0180] During the continuous pressure screening process, cell growth was observed. When all cells in the negative control wells died, the cell wells that had grown into cell clusters were resuspended in CHO medium containing the appropriate concentration of MSX, and then single-clonal cells were screened in 96-well plates using the limiting dilution method. After at least one subcloning, the cell lines were progressively expanded, identified, and then frozen in liquid nitrogen for preservation. The CHO-GS-KO cell lines that stably expressed PRRSV Gp4Fc, PRRSV Gp5Fc, or PRRSV VMFc proteins were named CHO-GS-KO-PRRSV Gp4Fc, CHO-GS-KO-PRRSV Gp5Fc, and CHO-GS-KO-PRRSSV VMFc, respectively.

[0181] Example 4: Identification of the CHO-GS-KO cell line expressing the fusion protein of porcine reproductive and respiratory syndrome virus (PRRSV) Gp4Fc, PRRSV Gp5Fc, and PRRSV MFc.

[0182] 1. Immunofluorescence assay (IFA) was used to detect the expression of PRRSV Gp4Fc protein, PRRSV Gp5Fc and PRRSV VMFc fusion protein.

[0183] CHO-GS-KO cell lines stably expressing PRRSV Gp4Fc, PRRSV Gp5Fc, or PRRSVFc proteins were selected and seeded in 24-well plates at a seeding density of 2 × 10⁶ cells / well. 4 Cells / mL were collected and incubated at 37°C in a 5% CO2 incubator to allow them to adhere and grow. When the cells reached approximately 60% confluence, the culture medium was discarded, and the cells were washed three times with PBS (pH 7.4) for 5 minutes each time. Cells were then fixed at -20°C for 30 minutes with a pre-cooled acetone and methanol mixture (1:1, volume ratio), followed by three washes with PBS for 5 minutes each time. Finally, cells were blocked with PBS containing 2% BSA at room temperature for 1 hour.

[0184] The FITC-labeled goat anti-pig IgG-Fc monoclonal antibody (1:1000 dilution) (Abcam) was incubated at 37°C in the dark for 1 hour. The cells were washed three times with PBS for 5 minutes each time. Working concentration of DAPI was added, and the mixture was incubated at 37°C for 10 minutes, followed by three washes with PBS for 5 minutes each time. The results were observed and photographed under an inverted fluorescence microscope. Figure 7 As shown, positive cells exhibit green fluorescence labeled with FITC.

[0185] 2. SDS-PAGE detection of purified PRRSV Gp4Fc, PRRSV Gp5Fc, or PRRSV MFc proteins

[0186] The CHO-GS-KO cell lines stably expressing PRRSV Gp4Fc, PRRSV Gp5Fc, or PRRSV MFc proteins were screened and cultured in 500 mL shake flasks until the cell density reached 4 × 10⁶ cells / year. 6 At a concentration of cells / mL, the cells were centrifuged at 4°C and 10,000 rpm for 10 min to collect the supernatant. After filtering the supernatant through a 0.22 μm filter, the supernatant was incubated overnight in nickel medium at 4°C. Then, the PRRSV Gp4Fc, PRRSV Gp5Fc, or PRRSV MFc proteins bound to the nickel medium were eluted and purified using an NGCQuest 10 chromatography system (BIO-RAD, USA). The eluted proteins were concentrated using a 30 kDa ultrafiltration tube, and 40 μL of the sample was then analyzed by SDS-PAGE. The results are shown below. Figure 8 As shown, a specific band appears at the target size.

[0187] 3. Identification of PRRSV p4Fc, PRRSV Gp5Fc, and PRRSV MFc protein dimers

[0188] Two 40 μL aliquots of recombinant CHO cell line expression samples were taken. One aliquot was treated with β-mercaptoethanol as a denaturing agent, while the other was not treated with β-mercaptoethanol. Western blotting was then performed following the steps outlined above. Results are as follows: Figure 9 As shown, the bands of Gp4Fc, Gp5Fc, and MFc proteins without denaturation agents are approximately twice the size of the bands with denaturation agents, indicating that proteins expressed by the fusion of Gp4, Gp5, or M with IgG3Fc form a stable dimer morphology under the action of disulfide bonds.

[0189] Example 5

[0190] Mouse immunization test of subunit vaccine

[0191] 1. Immunization program

[0192] Fifteen 6-week-old female BALB / c mice were selected and randomly divided into three vaccination groups (n=5 per group). Immunization was performed using a combination of different subunit vaccines (see Table 20). The vaccine was administered via intramuscular injection into the hind leg. The immunization schedule consisted of two doses, with a preferred dose of 200 μL per mouse, spaced three weeks apart. A booster immunization with the same dose was given three weeks after the initial immunization.

[0193] Table 20. Immunization experiments in mice by immunization method.

[0194]

[0195] 2. Indirect ELISA detection of mouse serum antibody levels based on PRRSVGp4Fc, PRRSVGp5Fc, or PRRSVFc proteins.

[0196] Serum samples were collected on days 0, 21, and 42 post-immunization. The titer of PRRSV-specific antibodies in mouse serum at different time points was detected using an indirect ELISA method to assess whether the subunit vaccine could effectively promote humoral immune responses. ELISA plates were coated with purified PRRSVGp4Fc, PRRSVGp5Fc, or PRRSVFc at 0.2 μg / well and incubated overnight at 4°C. After blocking with 5% BSA at 37°C for 1 h, and washing three times with PBST (5 min / wash), 100 μL of diluted test serum (1:1600) was added to each well, and the plates were incubated at 37°C for 1 h. After washing three times with PBST, 100 μL of diluted HRP-labeled goat anti-mouse IgG secondary antibody (1:10000) (Wuhan Antecjet Biotechnology Co., Ltd.) was added to each well. After washing three times with PBST, 100 μL of TMB substrate chromogenic solution (Beijing Solarbio Science & Technology Co., Ltd.) was added, and the plates were incubated at room temperature for 20 min. The reaction was terminated by adding 50 μL of 2 mol / L H2SO4 stop solution to each well, and the OD450 value was measured.

[0197] The results are as follows Figure 10 As shown in Figure A, compared to the PBS group, group A showed a significant increase in serum antibody levels against PRRSV p4Fc protein at 21 days post-immunization (P<0.05); compared to the PBS group, group B also showed a significant increase in serum antibody levels against PRRSV Gp4Fc protein at 21 days post-immunization (P<0.05). Furthermore, at 21 days post-immunization, the specific antibody level in group A was significantly higher than that in group B (P<0.05). At 42 days post-immunization, there was no significant difference in serum antibody levels against PRRSV p4Fc protein between groups A and B (P>0.05). No corresponding antibodies were detected in group C (the PBS control group) at days 0, 21, and 42 post-immunization. It is noteworthy that group A, despite not being immunized against the Gp4Fc antigen, still produced specific antibodies against the Gp4Fc protein, indicating that some serum antibodies were induced by the Fc antigen.

[0198] The results are as follows Figure 10As shown in Figure B, compared to the PBS group, group A showed a significant increase in serum antibody levels against PRRSV p5Fc protein at 21 days post-immunization (P<0.05); compared to the PBS group, group B showed a significant increase in serum antibody levels against PRRSV p5Fc protein at 21 days post-immunization (P<0.05). Furthermore, at 21 days post-immunization, the specific antibody level in group A was significantly higher than that in group B (P<0.05). At 42 days post-immunization, there was no significant difference in serum antibody levels against PRRSV p5Fc protein between groups A and B (P>0.05). No corresponding antibodies were detected in group C, the PBS control group, at days 0, 21, and 42 post-immunization.

[0199] The results are as follows Figure 10 As shown in Figure C, compared to the PBS group, group A showed a significant increase in serum antibody levels against PRRSVMFc protein at day 21 post-immunization (P<0.05); compared to the PBS group, group B also showed a significant increase in serum antibody levels against PRRSVMFc protein at day 21 post-immunization (P<0.05). Furthermore, at day 21 post-immunization, the specific antibody level in group A was significantly higher than that in group B (P<0.05). At day 42 post-immunization, there was no significant difference in serum antibody levels against PRRSVMFc protein between groups A and B. No corresponding antibodies were detected in group C (the PBS control group) at days 0, 21, and 42 post-immunization.

[0200] The above results indicate that the subunit vaccines prepared from the recombinant CHO-GS-KO cell line protein expressed in this invention can effectively induce the body to produce PRRSV-specific antibodies.

[0201] 3. Indirect ELISA detection of mouse serum antibody levels based on PRRSV-Q strain

[0202] The PRRSV-Q strain isolated and preserved in the laboratory was purified and coated onto ELISA plates at 0.4 μg / well, and incubated overnight at 4°C. Blocking was performed with 5% BSA at 37°C for 1 h. After washing three times with PBST (5 min / wash), 100 μL of diluted test serum (1:1600) was added to each well, and incubation was carried out at 37°C for 1 h. After washing three times with PBST, 100 μL of diluted HRP-labeled goat anti-mouse IgG secondary antibody (1:10000) (Wuhan Antejet Biotechnology Co., Ltd.) was added to each well. After washing three times with PBST, 100 μL of TMB substrate chromogenic solution (Beijing Solarbio Science & Technology Co., Ltd.) was added, and chromogenic reaction was carried out at room temperature for 20 min. The reaction was stopped by adding 50 μL of 2 mol / L H₂SO₄ stop solution to each well, and the OD450 value was measured.

[0203] The results are as follows Figure 11As shown, 21 days after immunization, antibody levels in groups A and B were significantly increased, while no antibodies against the PRRSV-Q strain were detected in group C, the PBS control group.

[0204] 4. Detection of PRRSV neutralizing antibody levels

[0205] (1) Inactivate the serum at 56℃ for 30 min. Serially dilute the inactivated serum with serum-free DMEM to a 1:256 concentration, and perform 3 replicates for each dilution.

[0206] (2) Add 50 μL of virus solution (100 TCID50) to each well. 50 After mixing, incubate at 37°C for 90 min, with virus control and cell control set up simultaneously;

[0207] (3) After 90 min, add 100 μL of Marc-145 cell suspension to each well, and then place the 96-well plate in a 37℃ 5% CO2 incubator for 72 h.

[0208] (4) After 72 hours, observe the cytopathic effect under a microscope and then calculate the serum neutralization titer. The maximum dilution of the serum that can completely inhibit the cytopathic effect is taken as the neutralization titer of the serum.

[0209] The results are as follows Figure 12 As shown, 21 days after the initial immunization, all vaccine-immunized groups exhibited neutralizing antibodies against PRRSV. At 42 days post-immunization, neutralizing antibody levels in groups A and B continued to rise, with group B showing a slightly higher level than group A. No neutralizing antibodies against PRRSV were detected in group C (PBS). These results indicate that combined immunization with PRRSV Gp4Fc, PRRSV Pp5Fc, and PRRSV MFc is more effective than combined immunization with PRRSV Gp5Fc and PRRSV MFc in increasing the production of neutralizing antibodies against PRRSV in mice.

[0210] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A combination of three fusion proteins characterized in that, The fusion protein is a Gp4Fc fusion protein, a Gp5Fc fusion protein and a MFc fusion protein. The amino acid sequence of the Gp4Fc fusion protein is shown as SEQ ID NO: 6; the amino acid sequence of the Gp5Fc fusion protein is shown as SEQ ID NO: 7; and the amino acid sequence of the MFc fusion protein is shown as SEQ ID NO:

8.

2. A gene encoding the combination of claim 1, characterized in that, The nucleotide sequence of the gene encoding the Gp4Fc fusion protein is shown as SEQ ID NO: 21; The nucleotide sequence of the gene encoding the Gp5Fc fusion protein is shown as SEQ ID NO: 22; The nucleotide sequence of the gene encoding the MFc fusion protein is shown as SEQ ID NO:

23.

3. A gene encoding the combination of claim 1, characterized in that, The nucleotide sequence of the gene is shown as SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO:

5.

4. A recombinant vector, characterized in that, The gene of claim 2 or 3 is inserted into the recombinant vector.

5. A recombinant cell line, characterized in that, The recombinant vector of claim 4 is included.

6. A method of constructing a recombinant cell line according to claim 5, wherein, The method comprises the following steps: The recombinant vector of claim 4 is packaged with a helper plasmid by a lentivirus to obtain a packaged lentivirus, which is introduced into a host cell to obtain a recombinant cell line.

7. The recombinant cell line constructed by the method of claim 6 is used for preparing a vaccine for preventing porcine reproductive and respiratory syndrome.

8. A porcine reproductive and respiratory syndrome virus subunit vaccine comprising an antigen and an adjuvant, characterized in that, The antigen comprises the combination of the three fusion proteins of claim 1.

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