Cloning And Expression Of The ORF2 Gene Of Porcine Circovirus Type Ⅱ In E.coli

Porcine circovirus type 2 (PCV-2) is the causative agent of postweaning multisystemic wasting syndrome (PMMS) that has become an emerging and important disease in swine. The ORF2 gene of PCV-2 is the main region encoding the structural protein that can be use to differentiate PCV-1 and PCV-2.Based on the published PCV-2 sequences, a pair of primers which were specific to the ORF2 genes of PCV-2 were designed. A 702-bp fragment was amplified by polymerase chain reaction(PCR). The DNA fragment was then successfully cloned into pGEM-T-Easy vector, which was confirmed by sequence analysis and restrict enzyme identification.Based on the analysis results of molecular software, Ten partition of the ORF2 of PCV-2 were cloned and inserted into the bacterial plasmid pGEX-4T-l. After identification by PCR and restricted enzyme, the constructed recombinant expressed plasmids was transformed into the receptive cells of E.coli ER2566 and induced by IPTG with a finial concentration of Immol/L, then the recombinant protein was highly expressed in the form of inclusion bodies. Western-blot analysis with the monoclonal antibody against the ORF2 of PCV-2 showed that nigh of the ten recombinant proteins shared good antigencity and specificity.After the inclusion bodies of recombinant proteins were purified with washing buffer consisting of 2mol/L, 4mol/L and 8mol/L urea for several times, the recombinant proteins were purified by electrodialysis method. Following the purification of the recombinant proteins, A indirect-ELISA was developed to detect the sera of piglets inoculated with PCV-2. The result indicated that six of the ten recombinant proteins have excellent reactogenicity.