PRRSV-1 virus-like particles and preparation method thereof
A PRRSV-1, virus-like technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, viral peptides, etc., can solve the problems of unsatisfactory vaccines, large-scale production, and complicated process of virus-like particles
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Embodiment 1
[0038] The design of the M gene sequence primer of embodiment 1 expression structural protein GP5 and matrix protein
[0039] Based on the published PRRSV LV strain (GenBank accession number: M96262.2) envelope structural protein GP5 and the nucleotide sequence of expression gene of matrix protein M, and design primers respectively for gene amplification; In matrix protein M gene The syn21 sequence is added to the upstream amplification primer as shown in SEQ ID NO.5, and the Flag sequence is added to the downstream amplification primer as shown in SEQ ID NO.6; the amplification primers are synthesized by Jilin Kumei Biotechnology Co., Ltd. The sequences and numbers of the primers for each gene amplification are as follows:
[0040] (1) The amplification primer sequence of GP5 gene is:
[0041] SEQ ID NO.1:
[0042] pGP5-F:5'-CTCGAGATGAGATGTTCTCACAAATTG-3';
[0043] SEQ ID NO.2: pGP5-R:5'-GGTACCCTAGGCCTCCCATTGCTC-3'; wherein, the 5'-end restriction site of the amplified pro...
Embodiment 2
[0050] Amplification and cloning of embodiment 2GP5 gene and M gene
[0051] Take 200 μL of PRRSV virus liquid, use the total RNA extraction kit to extract the total RNA in the virus liquid, and reverse transcribe it into cDNA. Using the cDNA as a template, using pGP5-F / pGP5-R and pM-F / pM-R in Example 1 as primers, set the reaction conditions according to the instructions of the gene synthesis report, and PCR amplify the GP5 gene and the M gene.
[0052] PCR products were separated and purified by 1% agarose gel electrophoresis. Mix 7 μL of the purified PCR product with 1 μL of pMD-19T Simple vector, 1 μL of T4 DNA ligase, and 1 μL of Buffer. In freshly thawed Trans5α competent cells, ice-bath for 20 minutes, heat shock at 42°C for 60 seconds, immediately ice-bath for 5 minutes, then activate at 37°C for 5 minutes, spread evenly on the LB culture plate containing ampicillin (50 μg / mL), 37 Cultivate upside down at ℃ for 12 hours, pick a single colony into LB liquid medium cont...
Embodiment 3
[0054] The construction of embodiment 3 recombination shuttle plasmids
[0055] The schematic diagram of the construction of the recombinant shuttle plasmid pFBD-GP5-M is as follows: figure 1 As indicated, the 19T-GP5 plasmid and the carrier plasmid were digested with restriction endonucleases XhoI and KpnI, and the GP5 gene was cloned into the downstream of the Pp10 promoter of the pFastBac Dual vector to construct the recombinant plasmid pFBD-GP5; the restriction endonuclease The 19T-M plasmid and the recombinant plasmid pFBD-GP5 were digested with enzymes BamHI and HindIII, the M gene was cloned downstream of the PpH promoter, and the recombinant shuttle plasmid pFBD-GP5-M was constructed and identified by enzyme digestion. The electrophoresis of pFBD-GP5-M is as follows figure 2 As shown, the recombinant plasmid construction and extraction methods are the same as in Example 2.
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