The Sequencing Of Porcine Parvovirus Genome And Prokaryotic Expression Of VP2

In order to determine the porcine parvovirus(PPV)gene sequence,7 pairs of specific primers overlaPPIng each other were designed and synthesized according to the complete PPV sequence(NC001718)in Genebank.The genome of RPBS2501 strain was amplified by PCR.The gene fragments were successfully cloned into the pEASY-Blunt vector and sequenced.The DNAStar software was used to assemble the sequencing results,and the nucleotide sequence of RPBS2501 strain with a length of 4946 bp was obtained.The complete coding region was obtained,and there was no base iNSertion or deletion in the sequence.Comparing the RPBS2501 strain sequences with the 12 PPV sequences published on Genebank,it was found that the homology of the RPBS2501 strain to the US isolated standard strain NADL-2(NC001718,5075bp)was as high as 99.8%.RPBS2501 strain NS gene nucleotide sequence isolated from China J-PPV strain(KF742500,5075bp),Hungarian isolated NADL-2 M1 strain(KF913345,5075bp),the United States isolated standard strain NADL-2(NC001718,5075bp)The homology is as high as 99.9%.The nucleotide sequence of VP2 protein of strain RPBS2501 was as high as 99.6% homology with GD2013 strain(KX242359,4723bp)isolated from China and the standard strain NADL-2(NC001718,5075bp)isolated from the United States.The VP2 gene sequence was codon optimized according to E.coli preference,and then the optimized PPV VP2 gene was synthesized,and the synthesized gene sequence was ligated to the pET-32a(+)vector.The coNStructed recombinant plasmid pET-32a-VP2 was identified by PCR and identified by double restriction enzyme digestion and traNSformed into E.Coil BL21.After induction,the molecular weight of the target band was found to be coNSistent with the expected size by SDS-PAGE.The purified target protein was identified by Western Blot,indicating that the protein was successfully expressed.This experiment successfully expressed and purified the VP2 protein,and laid the foundation for the development of VLPs with good reactogenicity and the development of safer and more effective vaccines.