Cloning, Sequencing And Expressing Of ORF2 Gene Of Porcine Circovirus Type 2 Isolated From Jilin Province

Porcine circovirus is a member of Circoviridae, which can be divided into two types, PCVI and PCV2. PCV2 can cause a disease named postweaning multisystemic wasting syndrome (PMWS), whereas PCVI is ubiquitous and nonpathogenic for pigs. PMWS has made a great loss in world's swine industry and aroused people's attention.Both PCVI and PCV2 were knowed that they contain 2 major open reading frames (ORF) that are ORF1 and ORF2. ORF1 encodes a putative protein involved in viral replication (Rep protein). Rep protein of PCV1 shares about 85% identity with that of PCV2, which is the major reason of PCVI and PCV2 have cross-reaction of antigenicity. ORF2-coding product is the major structural protein. This protein shares about 68% identity between two types virus; however, no cross-reaction of antigenicity is observed between them. Therefore, the ORF2 gene can be used to differentiate PCVI and PCV2.According to the nucleotide sequences of published porcine circovirus type 2 (PCV2) in GenBank, a pair of primers that were specific to the ORF2 gene of PCV2 were designed and synthesized in the research. ORF2 gene was amplified by polymerase chain reaction (PCR) from PCV2 Jilin strain(JLOl) which was isolated with PK-15 cell line before and had been characterised. The PCR product was cloned into the pMD-18T vector and then sequenced. The length of ORF2 gene is 702bp. By biology-software, the result of sequence analysis indicated that the identity is higher between strains isolated in our country than that in other countries.Using other pair of primers, the ORFs gene, a part of ORF2 gene, was amplfied by PCR. A fragment of 585bp of ORFs gene was obtained and cloned into pMD -18T vector and then transformed into pET-32a expression vector. The recombinant pET-ORFs plasmid was transformed into BL21 comptent cell of E.coli and induced by IPTG with a finial concentration of lmmol/L. SDS-PAGE analysis showed that the recombinant protein had been expressed. The protein could react with polyclonal antibody against PCV2 by Western-Blot test. The result declared that the expressing protein shared a good antigencity.