CN114134180A - Construction method of recombinant baculovirus expressing porcine reproductive and respiratory syndrome (GP) 5 protein - Google Patents
Construction method of recombinant baculovirus expressing porcine reproductive and respiratory syndrome (GP) 5 protein Download PDFInfo
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Abstract
The invention relates to the field of biological products for livestock, and particularly discloses a construction method of a recombinant baculovirus expressing a porcine reproductive and respiratory syndrome virus GP5 protein. The invention designs a synthetic primer according to the gene sequence of the Genbank blue ear virus CH-1a strain ORF5, amplifies ORF5 gene fragment and uses pFastBacTMUsing a Dual plasmid as a skeleton, inserting an ORF5 gene into the plasmid to obtain a baculovirus transfer vector pFBD-GP5, transforming the baculovirus transfer vector pFBD-GP5 into DH10Bac competent cells to obtain recombinant bacmid GP 5; sf9 insect cells were transfected to obtain recombinant baculovirus. The recombinant baculovirus is identified to be capable of efficiently expressing GP5 protein in insect cells, and the protein has good biological activity and immunogenicity.
Description
Technical Field
The invention relates to the field of biological products for livestock, in particular to a construction method of a recombinant baculovirus expressing a porcine reproductive and respiratory syndrome virus GP5 protein.
Background
Porcine Reproductive and Respiratory Syndrome (PRRS) is a highly contagious infectious disease caused by the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) that is primarily characterized by sow reproductive disorders and piglet respiratory symptoms. At present, the disease is widely prevalent in China, is mainly characterized by sow abortion, stillbirth, weak fetus, mummy fetus, difficult breathing, septicemia, high mortality and the like of piglets, has high morbidity, high fatality rate and low cure rate, and causes serious economic loss to the pig industry in China.
The PRRSV is a single-stranded positive-strand RNA virus with an envelope, the genome of the virus is not segmented, the total length of the virus is about 15kb, the virus is spherical or oval, the diameter of the virus is 45-65nm, and the virus is 20-face-body symmetric. The PRRSV genome contains 9 open reading frames which are respectively named as ORF1a, ORF1b, ORF2a, ORF2b and ORF3-ORF 7. Among them, ORF2a, ORF2b, ORF3-ORF7 encode structural proteins, and the encoded corresponding structural proteins are GP2, E, GP3, GP4, GP5, matrix protein (M) and nucleocapsid protein (N) in this order, and GP5, M and N are major structural proteins. The GP5 protein is an important envelope glycoprotein, has a molecular weight of 24-26kd, and contains 4 glycosylation sites. The protein contains a section of larger internal hydrophobic structural domain, is related to the anchoring effect of a membrane, and meanwhile, the GP5 protein is the protein with the largest variation among different strains, has both linear epitopes and conformational epitopes, can induce and generate neutralizing antibodies, and is the main immunogenic protein of PRRSV.
At present, the prevention and control of PRRS mainly takes immunization as the main part, and mainly takes vaccine immunization as the main part in China, and the commonly used vaccines comprise attenuated live vaccines and inactivated vaccines. The attenuated live vaccine has the advantages of stronger immunity, quick antibody generation, less dosage and low cost, but has the risks of toxin dispersion and strong virulence return. The inactivated vaccine has good safety, long time for inducing and generating the antibody, but high injection dosage and high cost. The development of subunit vaccines has therefore become a focus. Since GP5 protein contains a plurality of glycosylation sites, prokaryotic expression is easy to cause low protein activity. Baculovirus expression is a eukaryotic expression system, target protein can be processed and modified, and the biological activity of the target protein is better maintained, so that the GP5 protein is prepared by adopting the baculovirus expression system in the research.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention provides a construction method of the recombinant baculovirus which has a concise path and good immunoreactivity and expresses the GP5 protein of the porcine reproductive and respiratory syndrome virus.
The invention is realized by the following technical scheme:
a construction method of a recombinant baculovirus expressing a porcine reproductive and respiratory syndrome (GP) 5 protein comprises the following steps:
(1) amplification of GP5 gene and construction of recombinant donor plasmid:
according to the gene sequence of PRRSV CH-1a strain, a bioinformatics software is used for designing a primer, and ORF5 target genes are obtained through amplification; identifying the recombinant bacmid using a universal primer; using PRRSV CH-1a strain genome as a template, amplifying GP5 gene, and recovering and purifying a target band; the target fragment and a transfer vector pFastBacTMPerforming BamH I and Sal I double enzyme digestion on Dual respectively, recovering and purifying, connecting, chemically converting DH5 alpha competent cells, screening positive clones, extracting recombinant donor plasmid pFBD-GP5, and performing enzyme digestion sequencing identification;
(2) construction and identification of the GP5 recombinant shuttle plasmid:
transforming the donor plasmid pFBD-GP5 to a DH10Bac competent cell containing AcBacmid and Helper plasmids, culturing on a plate, screening, and selecting a white single colony; extracting DNA, carrying out PCR detection by using an M13 universal primer, screening a positive clone bacmid, named as rBacmid GP5, and extracting rBacmid-GP5 recombinant bacmid for cell transfection;
(3) obtaining and identifying GP5 recombinant baculovirus:
transfecting an Sf9 insect cell with a good growth state by adopting a liposome transfection kit and rBacmid-GP5, and collecting supernatant serving as a P0 generation recombinant virus after cytopathic effect is cultured; transmitting the cell supernatant to Sf9 insect cells for 3 generations, collecting the cell supernatant of the 3 rd generation, extracting virus DNA as a template, carrying out PCR detection by using a primer F, R for amplifying GP5 genes, and screening to obtain a positive recombinant virus named rBac-GP 5;
(4) western-blot identification of GP5 protein:
inoculating recombinant virus rBac-GP5 into Sf9 insect cells, and centrifugally collecting cell precipitates after infection; cell lysis solution PMSF is used for cell lysis and ultrasonic treatment, and supernatant is collected by centrifugation and used for Western-blot analysis;
(5) immunofluorescence identification of GP5 protein:
inoculating recombinant virus rBac-GP5 to Sf9 insect cells, washing with PBS after infection, and washing with PBS after methanol fixation; after blocking with NBS, PRRSV positive serum was diluted with PBS as primary antibody, incubated and washed with PBST; NBS diluted Anti-Swine IgG (H + L) was used as a secondary antibody, PBST was used for washing after incubation, and the result was observed under a fluorescence inverted microscope, and Sf9 cells were treated as a negative control.
The invention designs a synthetic primer according to the gene sequence of the Genbank blue ear virus CH-1a strain ORF5, amplifies ORF5 gene fragment and uses pFastBacTMUsing a Dual plasmid as a skeleton, inserting an ORF5 gene into the plasmid to obtain a baculovirus transfer vector pFBD-GP5, transforming the baculovirus transfer vector pFBD-GP5 into DH10Bac competent cells to obtain recombinant bacmid GP 5; sf9 insect cells were transfected to obtain recombinant baculovirus. The recombinant baculovirus is identified to be capable of efficiently expressing GP5 protein in insect cells, and the protein has good biological activity and immunogenicity.
The construction method further comprises the step (6) of identifying the immunogenicity of the GP5 protein:
inoculating recombinant virus rBac-GP5 into Sf9 insect cells, and centrifugally collecting cell precipitates after infection; cell lysis solution PMSF is used for cell lysis and ultrasonic treatment, and protein supernatant is collected through centrifugation; purifying the protein supernatant by column chromatography, mixing with water-in-oil-in-water adjuvant to prepare vaccine, immunizing 14-21 days old piglet, collecting blood at 14d, 28d and 56d after immunization, and detecting GP5 antibody.
Further preferably, in the step (1),
the upstream primer F is 5'-CGGATCCATGTTGGGGAAATGCTTGACC-3';
a downstream primer R: 5'-GCGTCGACCTAGAGACGACCCCATTGTT-3', respectively;
universal primer M13F: 5'-CCCAGTCACGACGTTGTAAAACG-3', respectively;
M13 R：5’-AGCGGATAACAATTTCACACAGG-3’。
further preferably, in step (2), white single colonies were selected by two rounds of blue-white screening by culturing on LB plates containing 50. mu.g/mL kanamycin, 7. mu.g/mL gentamicin, 10. mu.g/mL tetracycline, 100. mu.g/mL X-gal and 40. mu.g/mL IPTG for 48 hours.
Further preferably, in step (3), the transfected insect cells are cultured in a 28 ℃ incubator for 36-72h, and after cytopathic effect, the supernatant is collected as the recombinant virus P0 generation.
Preferably, in the step (4), after Sf9 insect cells are infected for 72 hours, cell precipitates are collected by centrifugation, the cells are lysed by PMSF cell lysate and subjected to ultrasonic treatment, and the supernatant is collected by centrifugation at 12000r/min for 10min and used for Western-blot analysis; the PRRSV positive serum was diluted 1:200 with antibody diluent as the primary antibody, HRP-labeled anti-porcine IgG was diluted 1:5000 with antibody diluent as the secondary antibody, and the normal Sf9 insect cell protein sample was used as the negative control.
Further preferably, in the step (5), after Sf9 insect cells are infected for 12h, PBS is used for washing for 3 times; pre-cooled methanol at 4 deg.C, fixing at 2-8 deg.C for 30min, and washing with PBS for 3 times; blocking with 5% NBS at 37 ℃ for 1 h; diluting PRRSV positive serum with PBS at a ratio of 1:200 to serve as a primary antibody, incubating for 1h at 37 ℃, and washing for 5 times with PBST; Anti-Swine IgG (H + L) diluted 1:100 with 5% NBS was used as a secondary antibody, incubated at 37 ℃ for 1H, washed 5 times with PBST, and observed under a fluorescence inverted microscope.
Further preferably, in the step (6), after Sf9 insect cells are infected for 72h, cell precipitates are collected by centrifugation; centrifuging at 12000r/min for 10min after ultrasonic treatment to collect protein supernatant; immunizing 14-21-day-old piglets with double negative blue ear antigen antibodies by the vaccine, and detecting GP5 antibodies by using a Haiboleblue ear ELISA antibody kit after blood collection.
The invention selects a eukaryotic expression system, namely an insect baculovirus expression system, can carry out post-translational processing modification on the protein, omits the complex processes of deformation, renaturation and the like of a prokaryotic system, displays the natural conformation of the target protein, and ensures that the expressed protein has good immunoreactivity, thereby laying a foundation and preparing materials for further researching and developing vaccines based on PRRSV VLPs.
Drawings
The invention will be further described with reference to the accompanying drawings.
FIG. 1 is a schematic flow diagram of a construction method of the present invention;
FIG. 2 is the amplification diagram of the gene sequence of GP5 of the CH-1a strain of porcine reproductive and respiratory syndrome virus;
in the figure: m represents the DNA molecular mass standard of DL 5000; 1-4 represent the amplification of the GP5 gene;
FIG. 3 is a PCR identification of the recombinant shuttle plasmid provided in the examples;
in the figure, M represents the DNA molecular mass standard of DL-5000; 1-10 represents PC of the recombinant shuttle plasmid;
FIG. 4 is a diagram showing the restriction enzyme identification of the recombinant donor plasmid provided in the example;
in the figure: m represents the DNA molecular mass standard of DL 5000; 1 represents pFBD-GP5 plasmid; 2 represents the double digestion product of pFBD-GP5 plasmid;
FIG. 5 is a blue and white screening of recombinant GP5 shuttle plasmid;
FIG. 6 shows CPE after infection of SF9 cells with recombinant infectious virus;
in the figure, A indicates that rBac-GP5 infects Sf9 cells; b indicates normal Sf9 cells;
FIG. 7 is an SDS-PAGE identification of the GP5 protein expressed by the recombinant baculovirus;
in the figure, M: protein Marker, 18-120 kd; 1: h5 cell control; 2: GP5 protein;
FIG. 8 is a graph showing the result of Western-bolt detection of baculovirus expressing GP5 protein;
in the figure, M: protein Marker, 10-120 kd; 1: BSA control, 2: GP5 protein;
FIG. 9 is an immunofluorescence map of recombinant baculovirus rBac-GP5 infected Sf9 cells;
FIG. 10 is a graph of ELISA antibody results after immunization of piglets with GP5 protein.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be exemplary only and are not intended to be limiting.
Aiming at the problems in the prior art, the invention provides a recombinant baculovirus expressing the GP5 protein of porcine reproductive and respiratory syndrome virus and a preparation method thereof, and the invention is described in detail below with reference to the accompanying drawings.
As shown in the attached figure 1, the method for expressing the GP5 protein of the porcine reproductive and respiratory syndrome virus by the baculovirus and the immunogenicity identification method thereof provided by the embodiment of the invention comprise the following steps:
(1) amplifying genes and constructing a recombinant donor plasmid;
(2) constructing and identifying a GP5 recombinant shuttle plasmid;
(3) obtaining and identifying recombinant baculovirus;
(4) identifying the expression of GP5 protein by Western-blot;
(5) indirect immunofluorescence identification of GP5 protein;
(6) and identifying the immunogenicity of the GP5 protein.
1. Materials and methods
1.1 materials
pFastBac Dual vector, PRRSV CH-1a strain, porcine reproductive and respiratory syndrome virus positive serum and Sf9 insect cells were stored in the laboratory, restriction enzymes were purchased from TaKaRa, competent E.coli DH5 alpha, DH10Bac were purchased from Shanghai Diego, insect cell culture Medium Sf-900TMIII SFM (1X) and transfection kit Cellffectin @ II Reagent were purchased from Gibco, HRP-labeled porcine secondary antibody, Anti-Swine IgG (H + L) from Sigma.
1.2 amplification of GP5 Gene and construction of recombinant Donor plasmid
According to the sequence of PRRSV CH-1a strain, a bioinformatics software is used for designing a primer.
The target gene amplification primers are as follows,
the upstream primer F is 5'-CGGATCCATGTTGGGGAAATGCTTGACC-3', and the upstream primer F is 5'-CGGATCCATGTTGGGGAAATGCTTGACC-3',
a downstream primer R: 5'-GCGTCGACCTAGAGACGACCCCATTGTT-3', respectively;
and (3) performing amplification by using PRRSV CH-1a strain accounting as a template to obtain a GP6 gene, and recovering and purifying a target band. And carrying out BamH I and Sal I double enzyme digestion on the purified target fragment and the pFastBac-Dual vector respectively, recovering, purifying, connecting, chemically transforming DH5 alpha competent cells, screening positive clones, extracting a recombinant donor plasmid pFBD-GP5, and carrying out enzyme digestion and sequencing identification.
1.3 construction and identification of the GP5 recombinant shuttle plasmid
Donor plasmid pFBD-GP5 was transformed into DH10Bac competent cells containing AcBacmid and Helper plasmids, cultured for 48h on LB plates containing kanamycin (50. mu.g/mL), gentamicin (7. mu.g/mL), tetracycline (10. mu.g/mL), X-gal (100. mu.g/mL) and IPTG (40. mu.g/mL), and white single colonies were selected for two rounds of blue-white spot screening. PCR detection is carried out by using M13 universal primers (M13-F: CCCAGTCACGACGTTGTAAAACG, M13-R: AGCGGATAACAATTTCACACAGG; and) positive clone bacmid is screened and named as rBacmid-GP5, and rBacmid-GP5 recombinant bacmid is extracted and used for cell transfection.
1.4 acquisition and characterization of recombinant baculovirus
Referring to the instruction of a liposome transfection kit Cellffectin II Reagent, rBacmid-GP5 is transfected into Sf9 insect cells with good growth state, the cells are cultured in a constant temperature incubator at 28 ℃ for 36-72h, and after cytopathic effect appears, the supernatant is collected to be used as P1 generation recombinant virus. And blind transferring the cell supernatant in Sf9 insect cells for 3 generations, collecting the cell supernatant of the 3 rd generation, extracting virus DNA as a template, carrying out PCR detection by using primers P1 and P2 for amplifying GP5 genes, and screening to obtain a positive recombinant virus named rBac-GP 5.
1.5 Western-blot identification of GP5 protein expression
Recombinant virus rBac-GP 5P 3 generation cytotoxicity is inoculated on Sf9 insect cells, and after 72 hours of infection, cell precipitation is collected by centrifugation. And (3) cracking cells by using PMSF (cell lysate) and carrying out ultrasonic treatment, centrifuging at 12000r/min for 10min, and collecting supernatant for Western-blot analysis. The PRRSV positive serum was diluted 1:200 with antibody diluent as the primary antibody, HRP-labeled anti-porcine IgG was diluted 1:5000 with antibody diluent as the secondary antibody, and the normal Sf9 insect cell protein sample was used as the negative control.
1.6 Indirect immunofluorescence identification of GP5 protein
Inoculating recombinant virus rBac-GP 5P 3 generation of cell virus to Sf9 insect cells with good growth condition, and washing 3 times with PBS after infecting for 12 hours; pre-cooled methanol at 4 deg.C, fixing at 2-8 deg.C for 30min, and washing with PBS for 3 times; blocking with 5% NBS at 37 ℃ for 1 h; diluting PRRSV positive serum with PBS 1:200 as a primary antibody, incubating for 1h at 37 ℃, and washing for 5 times with PBST; 5% NBS 1 Anti-Swine IgG (H + L) diluted 100 as secondary antibody, incubated 1H at 37 ℃, washed 5 times with PBST, observed under a fluorescence inverted microscope, and Sf9 cells treated as negative control.
1.7 animal immunization test
14-21 days old piglets (blue ear antibody antigen double negative) 15 heads were randomly divided into 3 groups, wherein group 1 was injected with vaccine prepared by rBac-GP5, group 2 was injected with supernatant of inactivated wild type baculovirus infected Sf9 insect cells, and group 3 was injected with PBS solution. Pre-and post-immunization 14d, 28d and 56d blood was collected and serum was isolated and blue-ear GP5 ELISA antibodies were determined.
2. Results
2.1 amplification of GP5 Gene and identification of recombinant donor plasmid pFBD-GP5
GP5 gene was amplified using PRRSV CH-1a genome as template and F, R as primers, respectively, to obtain a band of about 603bp (see FIG. 2), consistent with the expected size. Screening to obtain recombinant donor plasmid pFBD-GP5, carrying out double enzyme digestion verification of BamH I and SalI, and the result (shown in figure 3) shows that 2 specific bands are obtained after double enzyme digestion, wherein one band is GP5 gene about 603bp, and the other band is vector fragment about 6000 bp, and the two bands are consistent with the expected size. The pFBD-GP5 recombinant plasmid was further sequenced and verified, and the result showed that the inserted sequence was completely correct without base mutation.
2.2 obtaining recombinant shuttle plasmids and baculoviruses
The recombinant donor plasmid pFBD-GP5 was transformed into DH10Bac competent cells and screened for white single colony purification (see FIG. 4). Recombinant bacmid is extracted and PCR detection with M13 primer can amplify to one target band of 3100bp and the result is shown in FIG. 5. The recombinant Bacmid-GP5 liposome method is used for transfecting Sf9 cell insect cells, the insect cells are cultured at 28 ℃, the first generation has no obvious cytopathic effect, the obvious cytopathic effect appears after the insect cells are cultured for 72 hours in a blind mode from the 3 rd generation, the main manifestations are that the cells become bigger and become round, the boundary is transparent, the cell nucleus is obvious, and the result is shown in figure 6.
2.3 Western-blot identification of recombinant protein expression
Western-blot identification is carried out on the recombinant protein by a semidry method (see attached figures 7 and 8). The detection result shows that the recombinant virus infected Sf9 insect cell sample has a specific band of about 22KDa, and the band does not appear in normal cells. The GP5 gene of PRRSV is successfully expressed in Sf9 insect cells, and the expression product has certain reactogenicity.
2.4 Indirect immunofluorescence assay for recombinant protein expression
The rBac-GP5 baculovirus infects Sf9 insect cells, and after immunofluorescence detection, observation results under a fluorescence microscope show that the Sf9 cells infected by the rBac-GP5 show green fluorescence, while the normal SF9 cells do not show fluorescence, which indicates that the GP5 gene of PRRSV is correctly expressed in the Sf9 insect cells (see figure 9).
2.5 ELISA antibody detection
GP5 protein was used to immunize piglets and to detect ELISA antibodies in serum. The result shows that the serum GP5 antibodies of all groups of piglets before immunization are negative, which indicates that the test animals are normal; after immunization at 14d, the positive rate of the antibodies in the proteome is 100%, 28d reaches an extreme value, 56d is slightly reduced, and the antibodies of the piglets in the two control groups are negative, which indicates that the GP5 protein expressed by the recombinant baculovirus has good immunogenicity, and the result is shown in figure 10.
The PRRSV vaccines are divided into attenuated live vaccines and inactivated vaccines, and the two vaccines play a key role in the process of preventing and controlling the PRRS epidemic diseases. The commercial attenuated vaccines have various varieties and often have the problem of strong virulence or recombination with wild viruses; the whole virus inactivated vaccine has complex components and slow response speed; therefore, the development of a novel PRRSV vaccine with high efficiency and safety has important significance for effectively controlling and thoroughly eradicating the PRRSV. VLPs vaccines, which are self-assembled from viral structural proteins, are free of viral nucleic acids and can mediate efficient humoral and cellular immune responses, are currently considered safe and effective vaccine candidates.
The invention selects a eukaryotic expression system, namely an insect baculovirus expression system, can carry out post-translational processing modification on the protein, reduces the complex processes of denaturation, renaturation and the like of a prokaryotic system, displays the natural conformation of the target protein, and ensures that the expressed protein has good immunoreactivity, thereby laying a foundation and preparing materials for further researching and developing vaccines based on PRRSV VLPs.
The foregoing is considered as illustrative and not restrictive, and that any modifications, equivalents and improvements made within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (8)
1. A construction method of a recombinant baculovirus expressing a porcine reproductive and respiratory syndrome virus GP5 protein is characterized by comprising the following steps:
(1) amplification of GP5 gene and construction of recombinant donor plasmid:
according to the gene sequence of PRRSV CH-1a strain, a bioinformatics software is used for designing a primer, and ORF5 target genes are obtained through amplification; identifying the recombinant bacmid using a universal primer; using PRRSV CH-1a strain genome as a template, amplifying GP5 gene, and recovering and purifying a target band; the target fragment and a transfer vector pFastBacTMPerforming BamH I and Sal I double enzyme digestion on Dual respectively, recovering and purifying, connecting, chemically converting DH5 alpha competent cells, screening positive clones, extracting recombinant donor plasmid pFBD-GP5, and performing enzyme digestion sequencing identification;
(2) construction and identification of the GP5 recombinant shuttle plasmid:
transforming the donor plasmid pFBD-GP5 to a DH10Bac competent cell containing AcBacmid and Helper plasmids, culturing on a plate, screening, and selecting a white single colony; extracting DNA, carrying out PCR detection by using an M13 universal primer, screening a positive clone bacmid, named as rBacmid GP5, and extracting rBacmid-GP5 recombinant bacmid for cell transfection;
(3) obtaining and identifying GP5 recombinant baculovirus:
transfecting an Sf9 insect cell with a good growth state by adopting a liposome transfection kit and rBacmid-GP5, and collecting supernatant serving as a P0 generation recombinant virus after cytopathic effect is cultured; transmitting the cell supernatant to Sf9 insect cells for 3 generations, collecting the cell supernatant of the 3 rd generation, extracting virus DNA as a template, carrying out PCR detection by using a primer F, R for amplifying GP5 genes, and screening to obtain a positive recombinant virus named rBac-GP 5;
(4) western-blot identification of GP5 protein:
inoculating recombinant virus rBac-GP5 into Sf9 insect cells, and centrifugally collecting cell precipitates after infection; cell lysis solution PMSF is used for cell lysis and ultrasonic treatment, and supernatant is collected by centrifugation and used for Western-blot analysis;
(5) immunofluorescence identification of GP5 protein:
inoculating recombinant virus rBac-GP5 to Sf9 insect cells, washing with PBS after infection, and washing with PBS after methanol fixation; after blocking with NBS, PRRSV positive serum was diluted with PBS as primary antibody, incubated and washed with PBST; NBS diluted Anti-Swine IgG (H + L) was used as a secondary antibody, PBST was used for washing after incubation, and the result was observed under a fluorescence inverted microscope, and Sf9 cells were treated as a negative control.
2. The method of construction of claim 1, wherein: further comprises the step (6) of identifying the immunogenicity of the GP5 protein: inoculating recombinant virus rBac-GP5 into Sf9 insect cells, and centrifugally collecting cell precipitates after infection; cell lysis solution PMSF is used for cell lysis and ultrasonic treatment, and protein supernatant is collected through centrifugation; purifying the protein supernatant by column chromatography, mixing with water-in-oil-in-water adjuvant to prepare vaccine, immunizing 14-21 days old piglet, collecting blood at 14d, 28d and 56d after immunization, and detecting GP5 antibody.
3. The method of construction of claim 1, wherein: in the step (1), an upstream primer F: 5'-CGGATCCATGTTGGGGAAATGCTTGACC-3'; a downstream primer R: 5'-GCGTCGACCTAGAGACGACCCCATTGTT-3', respectively; universal primer M13F: 5'-CCCAGTCACGACGTTGTAAAACG-3', respectively; M13R: 5'-AGCGGATAACAATTTCACACAGG-3' are provided.
4. The method of construction of claim 1, wherein: in step (2), the culture was carried out for 48 hours on LB plates containing 50. mu.g/mL kanamycin, 7. mu.g/mLl gentamicin, 10. mu.g/mL tetracycline, 100. mu.g/mL X-gal and 40. mu.g/mL IPTG, two rounds of blue-white spot screening were carried out, and white single colonies were selected.
5. The method of construction of claim 1, wherein: in the step (3), the transfected insect cells are cultured in a constant temperature incubator at 28 ℃ for 36-72h, and after cytopathic effect appears, the supernatant is collected to be used as the P0 generation recombinant virus.
6. The method of construction of claim 1, wherein: in the step (4), after Sf9 insect cells are infected for 72 hours, cell precipitates are collected by centrifugation, the cells are lysed by PMSF lysate and subjected to ultrasonic treatment, and the cells are centrifuged at 12000r/min for 10min to collect supernatant for Western-blot analysis; the PRRSV positive serum was diluted 1:200 with antibody diluent as the primary antibody, HRP-labeled anti-porcine IgG was diluted 1:5000 with antibody diluent as the secondary antibody, and the normal Sf9 insect cell protein sample was used as the negative control.
7. The method of construction of claim 1, wherein: in the step (5), Sf9 insect cells are washed 3 times by PBS after being infected for 12 h; pre-cooled methanol at 4 deg.C, fixing at 2-8 deg.C for 30min, and washing with PBS for 3 times; blocking with 5% NBS at 37 ℃ for 1 h; diluting PRRSV positive serum with PBS at a ratio of 1:200 to serve as a primary antibody, incubating for 1h at 37 ℃, and washing for 5 times with PBST; Anti-Swine IgG (H + L) diluted 1:100 with 5% NBS was used as a secondary antibody, incubated at 37 ℃ for 1H, washed 5 times with PBST, and observed under a fluorescence inverted microscope.
8. The method of construction of claim 2, wherein: in the step (6), after Sf9 insect cells are infected for 72h, centrifuging and collecting cell precipitates; centrifuging at 12000r/min for 10min after ultrasonic treatment to collect protein supernatant; immunizing 14-21-day-old piglets with double negative blue ear antigen antibodies by the vaccine, and detecting GP5 antibodies by using a Haiboleblue ear ELISA antibody kit after blood collection.
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