Cloning Structural Protein P32 Gene Of Capripoxvirus And Its Expression In Escherichia Coli

Capripoxvirus causes a severe and highly contagious disease in sheep or goats,Capripox is classified as group A by Office International des Epizooties (OIE) and group first diseases in China. Capripoxvirus is the members of the capripoxvirus genus of the chordopoxvirnae of the poxvirdae. P32 protein of capripoxvirus is one of the virus structural protein which is presently isolated and identified in all capripoxvirus strains in countries.It stands out on the virus envelope surface and contains neutralized antigen epitope.The P32 gene of capripoxvirus was amplified by PCR using specific primers.The product of PCR was inserted to the plasmid pMD18-T.Then the P32 gene from the identified recombinant was amplified by PCR with another pair of primers containing EcoRâ… and Xhoâ… sites and digested with EcoRâ… and Xhoâ… .The expression vector pET-30a and pGEX-4T-1 were digested by EcoRâ… and Xhoâ… ,respectively.The target gene P32 was subcloned into vector pET-30a and pGEX-4T-1.Positive clones named as pET-P32 and pGEX-P32 with target gene were identificated by restriction analysis , PCR and DNA sequencing .The two recombinant pET-P32 and pGEX-P32 were expressed in Escherichia coli induced by IPTG .The molecular weights of expressed protein were about 37.5ku (poly-His fusion) and 57ku (GST fusion ),respectively. The results of Western-blotting showed that the P32 protein could be detected by the capripox positive serum . It indicated that the P32 protein be of immunoreactivity...