Cloning And Expression Of The P32 Gene Of Capripoxvirus And Establishing PCR Diagnostic Method

Capripox (Sheeppox and goatpox),caused by capripoxvirus,is an acute feverish and contagious disease in sheep or goat.Capripox is classfied as group A diseases by Office International des Epizooties (OIE)and group first diseases in China.P32 protein is one of the virus structure proteins.It stands out on the virus envelope surface and contains neutralized antigen epitope.The viral attachment protein gene is specific for capripoxvirus.The P32 gene of capripoxvirus were amplified by PCR.The product of PCR was cloned to the plasmid pGEM-T-Easy.Then the P32 gene was disparted to three parts.Three parts of P32 gene were inserted into the expressed plasmids pGEX-4T-1, which were identified by PCR and restriction enzyme analysis.The recombinant plasmids of pGEX-4T-1-N2/3, pGEX-4T-1-N1/3, pGEX-4T-1-M werem successfully constructed-Three parts of P32 gene were expressed in E.coli induced by IPTG in the form of inclusion bodied.The molecular weights of expressed protein are 52KDa,38KDa and 40KDa respectively.The expressed proteins were purified by electrodialysis and detected with ELISA .The results of ELISA demonstrated that the three expressed proteins shared reaction with standard positive serum of capripox.According to the attachment gene sequences of the Capripoxvirus, primers were designed. A 192bp target gene was amplified by PCR.The target gene was part of the attachment gene of capripoxvirus by sequence analysis.Based on the optimized experiments of PCR,the specifity and sentitivity of this diagnostic method were tested.The diagnostic PCR method was primarily established for capripoxvirus.