Preparation method, vaccine composition and application of a porcine pseudorabies virus subunit vaccine
A technology for porcine pseudorabies and vaccine compositions, which is applied in the field of preparation methods and vaccine compositions prepared therefrom, and can solve the problems of application limitation, high production cost, less immunogenicity than attenuated vaccines and inactivated vaccines, etc.
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Embodiment 1
[0059] Example 1 Tandem expression of porcine pseudorabies virus gB protein fragment and gD protein
[0060] 1. Amplification of porcine pseudorabies virus gB protein fragment
[0061] The well-grown PK15 cells were inoculated with PRV HN1201 virus, 200 μL of the harvested virus solution was taken, and PRV genomic DNA was extracted according to the instructions of Geneaid's viral nucleic acid extraction kit II kit. Use primers gBF1 and gBR1 to extend gB gene 62-148aa, use primers gBF2 and gBR2 to extend gB gene 546-700aa, use Overlapping PCR to amplify the two together, and use primer design to add GGSG linking amino acids between the two. The primers are shown in Table 1, the PCR system is shown in Table 2, and the PCR reaction conditions are shown in Table 3.
[0062] Table 1 Primer for gB protein fragment amplification
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[0064] Table 2 PCR system
[0065] 2×PrimeSTAR GC buffer 25μL PRV Genomic DNA 1μL primers(10pM) 1μL / 1μL dNTPs (2.5mM) 4μL PrimeSTAR(2.5U / μL) 0.5μ...
Embodiment 2
[0086] Example 2 Preparation of porcine pseudorabies virus gB protein
[0087] 1. Amplification of porcine pseudorabies virus gB gene
[0088] Using the PRV genomic DNA extracted in Example 1 as a template, primers gBF and gBR were used to amplify HNgB gene 62-752aa. The primers are shown in Table 6, the PCR system is in accordance with Table 2, and the reaction conditions are in accordance with Table 3.
[0089] Table 6 gB gene amplification primers
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[0092] 2. Construction of the donor plasmid
[0093] With reference to the construction method of the donor plasmid in Example 1, the PCR product amplified in step 1 was recovered to construct the donor plasmid, which was correctly named pFastBac-HNgB after identification.
[0094] 3. Construction of recombinant Bacmid
[0095] With reference to the construction method of recombinant Bacmid in Example 1, the donor plasmid obtained in step 2 was constructed to construct recombinant Bacmid, and the correct recombinant Bacmi...
Embodiment 3
[0100] Example 3 Preparation of porcine pseudorabies virus gD protein
[0101] 1. Amplification of porcine pseudorabies virus gD gene
[0102] Using the PRV genomic DNA extracted in Example 1 as a template, the gD gene was amplified using the primers gD18F and gD353R in Table 4 in Example 1. The PCR system is based on Table 2, and the reaction conditions are based on Table 3.
[0103] 2. Construction of the donor plasmid
[0104] With reference to the construction method of the donor plasmid in Example 1, the PCR product amplified in step 1 was recovered to construct the donor plasmid, which was correctly named pFastBac-HNgD after identification.
[0105] 3. Construction of recombinant Bacmid
[0106] Referring to the construction method of recombinant Bacmid in Example 1, the donor plasmid obtained in step 2 was constructed to construct recombinant Bacmid, and the correct recombinant Bacmid was identified as Bac-HNgD.
[0107] 4. Acquisition and passage of recombinant baculovirus
[0108...
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