Cloning And Expression Of CPV P32 Gene In E.coli And Establishment Of Multi-PCR

Capripoxvirus causes a severe and highly contagious disease in sheep and 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 chordopoxvirinae of the poxvirdae. P32 protein stands out on the virus envelope surface and contains neutralized antigen epitope. It is one of the virus structural protein which is presently isolated and identified in all capripoxvirus strains in countries.According to the different gene sequences of the Capripoxvirus, two pairs of primer were designed. 289bp and 969bp target fragments were amplified by multi-PCR. In order to establish a rapid, sensitive and special multi-PCR method, optimized the proportion of the primers, dNTP and condition of the reaction. By using the method to detect the CPV material and orf. virus, the method was proved special. And the method has high sensitivity that could detect capripoxvirus from 10(-7) diluted virus. The method could apply for the quickly diagnosis of CPV.The P32 gene of capripoxvirus was amplified by PCR. The product of PCR was cloned to the plasmid pMD 18-T. Then the P32 gene was inserted into the expressed plasmid pGEX-4T-1, which was identified by PCR, restriction analysis and DNA sequencing. The recombinant pGEX-P32 was expressed in Escherichia coli induced by IPTG. The molecular weight of expressed protein was about 58ku. 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. The expressed protein was purified by electrodialysis and detected with ELISA. The results of ELISA demonstrated that the expressed protein shared reaction with standard positive serum of capripox.