Construction Of Infectious Clone Of Porcine Circovirus Type2 And Cloning And Expression Of ORF2

Porcine cicovirus (PCV) is one of the smallest viruses in animals so far. Two types of PCVs have been recognized to date, PCV1 and PCV2, Which are both classified in the Circoviridae family. Porine circovrus type 2 is suspected to be a kind of potential pathogen of PMWS(postweaning multisystemic wasting syndrome).PMWS caused huge economic loss on world pig industry, and PCV2 becomes a lot splt. In the study, we cloned the complete genome of porcine circovirus type 2 from lymph node of pigs with PMWS and this complete genome of porcine circovirus type 2 was sequenced. The infectious clone of porcine circovirus type 2 was then constructed and indentified. Meanwhile, we cloned the gene of porcine circovirus type2 ORF2 and expressed this coding production in E.coli successfully.This study included two parts.PartⅠ: Cloning and sequencing of complete genome of porcine circovirus type 2 (PCV-2) SDZQ-1 strains.According to the published genomic sequence of porcine circovirus type 2,a pair of specific primers was designed to amplify the complete genome of porcine circovirus type 2 from lymph node of piglets with PMWS, and this amplified production was cloned into pMD-18-T vector and suquenced. The result of sequencing showed the genomes of porcine circovirus type 2 were 1767bp in length. This sequence were designated SDZQ-1. This sequence was compared with other published genomes of porcine circovirus type 2 by DNAstar. The results shows that the sequences of SDZQ-1 were closely related to those of other porcine circovirus type 2 in the GenBank, and homology of porcine circovirus type 2 nucleotide sequence were 95.1% to 99.1%. And homology of ORF1 and ORF2 amino acid sequence were also high, ranging from 96.2 % to99.7% and from 91.0% to 98.7% respectively.PartⅡ: Construction of the chimeric infectious DNA clone of porcine circovirus type2. homology in complete nucleotide sequence compare with others. The two complete genome were connected into PUC19 subsequently in norientation to construct doubt copy plsmid pUC2PCV2. Transfect PK-15 cells with pUC2PCV2 in vitro.After four passages, the infectivity of the cloned chimeric PCV-2 genome was confirmed by PCR and IFA.PartШ:Expressing of ORF2 gene of porcine circovirus type 2 of SDZQ-1Using other pair of primers, the ORFs gene, a part of ORF2 gene, was amplfied by PCR. Afragment 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 Wastransformed 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 resultdeclared that the expressing protein shared a good antigencity.