Prokaryotic Expression And Identification Ofcanine Parvovirus VP2 Gene

Canine parvovirus(CPV) VP2 protein is the main structural protein of CPV,accounting for about 90% of the capsid proteins,encoding the main antigenic determinant.It is not only the main immune protective protein,but also a good diagnostic antigen.Therefore,expression and identification of VP2 protein,is of great significance for the development of specific diagnostic antigen,meanwhile,providing datas for studying canine parvovirus popular strains in Changchun City.In this study,the sample was the dogs’ feces which were from a animal hospital in Chang chun City.According to the CPV-VP2 gene sequences from GenBank,a pair of specific primers was designed that was used to amplify a 1790 bp genetic segment.The fragment was cloned to the pMD18-T vector,constructing the recombinant cloning vector pMD18-T-VP2.The result showed that VP2 gene was successfully amplified after double-enzyme cleavageand and gene sequencing analysis.Through analysing the sequence,the nucleotides of VP2 gene had a similarity to 99.2%~99.9% compared to ten countries’ from GenBank,amino acid reached 99.0%~ 99.8%,consequently,the genetic segment was identified as CPV-VP2.It had a close relative of a strain GQ379042.1,which is new CPV-2a,only one base was different between them,so it was belong to new CPV-2a and GenBank accession number was applied,it was KP686093.Then,the VP2 gene was subcloned into the prokaryotic expression vector pET-32 a,the recombinant expression vector pET-32a-VP2 was identified after double-enzyme cleavageand,PCR and sequencing.The results showed that the VP2 gene was successfully cloned into pET-32 a vector, the ORF and the sequence were correct.Therefore,the recombinant expression vector pET-32a-VP2 was constructed successfully.Transforming the recombinant expression vector pET-32a-VP2 into E.coli BL21(DE3) competent cells,inducing protein expression of VP2 gene.The results showed that a distinct protein band about 87 kD was appeared and the protein was expressed in the form of inclusion body. In addition,through optimizing the conditions of induction,a conclusion was sobering —IPTG was 2mM,time was 4 hours,temperature was 37℃,the number of expressing protein was the largest.Then,protein was purified by nickel column.Last,the VP2 protein had a reaction activity indicated by Western blotting.