Research On Construction Of Infectious Porcine Circovirus Type 2 Molecular Clone And The Expression Of ORF1 Gene

Porcine Circovirus(PCV) is a small, nonenveloped, icosahedral mammalian virus containing a circular single-stranded DNA genome, which now has been assigned to the Circoviridae family. PCV-lwas first described in 1974 by Tisher as a picornavirus-like contaminant of the permanent pig kidney cell culture PK-15(ATCC-CCL33) and accepted to be a nonpathogemic virus. However, PCV-2 has been associated with postweaning multisystemic wasting syndrome(PMWS).In this research ,a pair of specific primers was designed according to the published genome sequences of PCV-2 to amplify the full length of viral genome by PCR from three samples with clinical signs of PMWS. The PCR product was cloned into pMD-18T vector and sequenced. After comparison the sequence with other PCV strains in GenBank. phylogenetic analysis was performed on the PCV strains. The results showed that there was 95.7%-99.2% nucleotide identity among the three strains and more than 95.1% identity between our cloned genomes and other PCV-2 strains in GenBank. The ORF1 of the cloned genomes shared more than 97.8% nucleotide identities and more than 98.0% deduced amino acid identities with other PCV-2 isolates, while the ORF2 has 90%-98% nucleotide sequence identity and 87%-99.1% amino acid identity. Phylogenetic tree analysis showed there is correlation between PCV-2 isolates and geographic regions. The circulated genomes are transfected into PK-15 cell line with Ipofectamine?2000. IFA was conducted after 3 passages of the transfected cells and the results showed that the genome of QD strain was infectious.The PCV-2 genomic DNA was digested with Sac II and then ligated to form concatemers ,. the concatemers was cloned into pBluescript SK + vector to construct the molecular PCV-2 clone: pSK2PCVG. The molecular PCV-2 clone was transfected into PK-15 cells by Ipofectamine mediated transformation., IFA showed that pSK2PCVG was infectious in vitro.In another research, a pair of specific primers were designed and synthesized according to the sequence of ORF1 gene of PCV-2 QD strain.The complete DNA fragment of ORF1 gene was obtained by PCR. Its nuclectide sequence was determined by the dideoxy-mediated chain termination method. Sequence analusis showed that the complete open reading frame (ORF) of ORF1 gene encoding 314 amino acid was 945bp in length. Then, the DNA fragment of ORF 1 gene was subcloned into prokaryotic expression vectorpET-28a and pGEX-KG, the specific non-fusion and fusion protein with GST of molecular weight 38kD and 63kD was expressed in E.coli BL21(DE3).Western blotting assay indicated that the polyclonal antibody against PCV-2 could recognize these two proteins.In this study, three complete genomes of PCV-2 were amplified and sequenced. Phylogenetic tree analysis showed there is correlation between PCV-2 isolates and geographic regions. Consequently, We construct the infectious molecular clone of porcine circovirus type 2. Moreover, ORF1 gene was expressed in E.coli. This study will give some help on studying epidemiology, vaccine , diagnosis and control of PCV-2 in China.