Gene Analysis Of Porcine Bocavirus Type 2 GZJS2018 Strain And Prokaryotic Expression Of VP2 Protein

Porcine Bocavirus(PBoV)is a member of the family Parvoviridae,subfamily Parvoviridae and genus bocavirus.Bocavirus was discovered as early as the early 1960s,but it was only detected in Swedish piglets suffering from weaning multiple system failure syndromes in 2009.At present,there are relatively few reports on porcine bocavirus.Porcine Bocavirus mainly causes diarrhearespiratory diseases and postweaning multisystemic wasting syndrome.In recent years,although porcine bocavirus has made certain research progress in the amplification of the whole gene sequence,detection methods,epidemiological investigations,etc.But its research on isolation and culture of cells?genotyping?pathogenic mechanism is not thorough enough.Moreover,there is no new progress in the study of the hairpin structure at both ends of the porcine bocavirus genome,so the research of porcine bocavirus is still lacking in many aspects.In recent years,various domestic provinces have successively reported relevant researches on the porcine bocavirus genome sequence,but there have not been any research reports on the porcine bocavirus and its genome in Guizhou Province.In this study,the genome sequence of the porcine bocavirus GZJS2018 strain in Guizhou Province was determined,and its biological information was analyzed using related software,and the prokaryotic expression study of part of the VP2 gene of the porcine bocavirus GZJS2018 strain was carried out for PBoV serum,it lay a foundation for serological detection method and vaccine development.1 Gene analysis of Porcine Bocavirus type 2 GZJS2018 strainIn order to obtain the Porcine Bocavirus 2(PBoV2)genome sequence,four pairs of specific primers were designed by comparing the complete gene sequence of porcine bocavirus included in Gen Bank and combining with the characteristics of each gene.After nucleic acid was extracted from fecal samples which were screened as positive for porcine bocavirus type 2,different overlapping genome fragments of the virus were amplified by PCR,cloned and sequenced respectively,and spliced to obtain an approximate full-length5134 bp gene sequence(PBoV GZJS2018).The genome has three open reading frames ORF1 ? ORF2 ? ORF3,which encode four viral proteins,NS1 ? VP1/VP2 ? and NP1,respectively.Using DNAstar software to analyze its nucleotide homology,it was found that the similarity between the PBoV GZJS2018 strain and the PBoV G2 gene group was the highest,ranging from 91.1% to 93.4%.Among them,the highest nucleotide homology of the PBoV GZJS2018 strain with the PBoV type 2 ZJD strain(accession number: HM053694.2)is 93.4%.The results of the genetic evolution analysis system showed that the PBoV GZJS2018 strain and the PBoV G2 gene group are in the same large branch,and the PBoV type 2 ZJD strain(accession number: HM053694.2)is in the same small branch,so the relationship is closest.The soluble,transmembrane region,secondary structure,tertiary structure and epitope prediction analysis results of the four viral proteins of the PBoV GZJS2018 strain NS1?VP1?VP2?NP1 and VP2 protein showed that the four proteins of PBoV GZJS2018 are all soluble proteins.And there is no transmembrane region and signal peptide,the secondary structure is mainly random coils,and the VP2 protein also has multiple B cell epitopes.2 Prokaryotic expression of partial gene protein of Porcine Bocavirus type2 GZJS2018 strain VP2In this experiment,based on the failure of the VP2 gene expression in the previous stage,through the analysis of the VP2 gene B cell epitope,the VP2 gene with relatively concentrated in 1 ? 744 bp epitope was selected from the VP2 gene(VP2~*)prokaryotic expression research.In the experiment,specific primers were used to amplify the VP2~* gene and cloned into pCIone 007 Versatile Simple Vector,and then subcloned into the prokaryotic expression vector pET-32a(+);finally,identified the constructed pET-32a-VP2~* plasmid,they were transferred to BL21(DE3)competent cells for inducible expression studies.The results showed that the optimal expression conditions for the pET-32a-VP2~* prokaryotic expression plasmid constructed were 37? 1.0 mmol/L IPTG induction for 2 h;SDS-PAGE electrophoresis showed that the obtained fusion expression protein was accordantly with the expected size,the fusion protein is approximately 47 KDa;after transferred the fusion protein to the PVDF membrane,the His-tag monoclonal antibody is used as the primary antibody,and the HRP goat anti-rabbit Ig G is used as the secondary antibody.Western-blotting detection is performed,and a blue bar of the expected size can be seen the band indicates that the PBoV GZJS2018 strain VP2~* prokaryotic expression plasmid was successfully constructed.