Isolation,identification Of CPV And Prokaryotic Expressionof The VP2 Gene

Canine parvovirus disease is a highly contagious and intense infectious disease caused by canine parvovirus(CPV)in dogs.It is mainly characterized by enteritis and myocarditis.The disease is widely distributed throughout the world.In recent years,the incidence of disease has been increasing year by year,which has brought great economic losses to the dog industry.VP2 protein is the main structural protein of CPV and the main antigenic determinant of CPV.In this study,a strain of CPV was isolated from the stool of a canine parvovirus disease,which was identified and prokaryotic expression of the VP2 gene.According to the CPV gene sequence published by GenBank,1 pair was used to identify CPV primers,and the target fragment size was 1755 bp.The reference design was used to identify canine coronavirus(CCV),canine distemper virus(CDV),and canine parainfluenza virus(CPIV).Primers were used to detect whether the infected dog was infected with other viruses;half of the cell infections(TCID50)were determined by immunofluorescence assay.The results showed that the gene fragment amplified by the primers used to identify CPV was consistent with the expected size;the genetic and evolutionary analysis of the target gene and CPV-VP2 gene in GenBank showed that this gene and new CPV-2a strain GQ379042.1 has the highest homology,thus confirming that the isolated virus is CPV and is subtype new 2a;other virus detection results indicate that CCV,CDV,and CPIV are negative;when 5 generations of blind transmission,the TCID50 is 1040/mL.The VP2 gene of the strain was ligated into the prokaryotic expression vector pET-32a,and the recombinant expression plasmid pET-32a-VP2 was obtained,and the correct prokaryotic expression vector pET-32a-VP2 was transformed into competent cell Kcoli BL21(DE3)induces expression.The results showed that the expression of the target protein was an inclusion body precipitate with a size of about 87 kDa.The expression of the target protein was the highest when the IPTG concentration was 1 mM,the induction time was 4 h,and the induction temperature was 32?.The expression of the VP2 protein was confirmed to be reactive by Western blot.