Cloning and Prokaryotic Expression of ORF1 Gene of Porcine Circovirus Type 2

In order to provide a scientific basis for the diagnosis and prevention of porcine circovirus disease,ORF1 gene of porcine circovirus type 2 (PCV2) GZ strain was obtained by PCR with specific primers designed according to PCV2 sequence in GenBank. Amplification product was connected with pMD18-T vector. After the identification, subclone it into pET-30a(+) prokaryotic expression vector. It was sequenced after double enzyme identification.The results showed that the purpose fragment of ORF1 gene of PCV2 was in the right position of pET-30a(+) vector,and we successfully constructed pET30a-PCV2-ORF1 prokaryotic expression vector.The recombintant plasmid was transformed into Rosetta, expressed by IPTG induction, and detected by SDS-PAGE.It showed that PCV2-ORF1 had efficient fusion expression in the pET-30a(+) and the expressed protein was 42 ku.The expression products, which existed mainly as inclusion bodies,were purified by Ni²⁺-affinity chromatographic column.Western blotting analysis showed that the purified products could react with anti-His MAb.