Construction Of Infectious Clone Of Porcine Circovirus Type 2 And Cloning And Expression Of ORF1

Porcine cicovirus (PCV) is one of the smallest viruses in animals so far. Two typesof PCVs have been recognized to date, PCV 1 and PCV 2, which are both classified inthe Circoviridae family. Porcine circovirus type 2 is suspected to be a kind of potentialpathogen of PMWS (postweaning multisystemic wasting syndrome). PMWS causedhuge economic loss on world pig industry, and PCV2 becomes a hot spot. In this study,we cloned the complete genome of porcine circovirus type 2 from lymph node of pigswith PMWS and this complete genome of porcine circovirus type 2 was sequenced. Theinfectious clone of porcine circovirus type 2 was then constructed and identified.Meanwhile, we cloned the gene of porcine circovirus type 2 ORF1 and expressed thiscoding production in E.coli successfully.According to the published genomic sequence of porcine circovirus type 2, a pairof specific primers was designed to amplify the complete genome of porcine circovirustype 2 from lymph node of piglets with PMWS, and this amplified production wascloned into pGEM-T easy vector and sequenced. The result of sequencing showed thegenomes of porcine circovirus type 2 were 1767bp in length. This sequence weredesignated ZHEJIANG2006 (GenBank accession EF210106) . This sequence wascompared with other published genomes of porcine circovirus type 2 by DNAstar. Theresults showed that the sequences of ZHEJIANG2006 were closely related to those ofother porcine circovirus type 2 in the GenBank, and homology of porcine circovirustype 2 nucleotide sequence were 95．1% to 99．1%. And homology of ORF1 and ORF2amino acid sequence were also high, ranging from 96．2% to 99．7% and from 91．0% to98．7% respectively.We mutated two nucleotide positions 1162 (T1162G) and 1164 (C1164T) from theZHEJIANG2006 sequence using overlap extension method. The mutations of T1162Gand C1164T made a glycine changing to a serine in the amino acid sequence encoded byORF2．The mutant sequence were inserted into pBluescriptⅡSK(+) expression vectors,and constructed the recombinant plasmid pSK-PCV2 (mut). Then, the 1767bp completed sequence of PCV2 (mut) was excised from the recombinant plasmidpSK-PCV2 (mut) by digestion with the Sac I restriction enzyme. The digested genomicDNA was purified and then the circular genomic DNA was generated by self-ligatingwith T4 DNA ligase in vitro. PK-15 cells were transfected with the PCV2 (mut) circulargenome using Lipofectin Reagent. PK-15 cells transfected with the pSK-PCV2 (mut)regarded as negative control. After several continuous passages, the expression of thecircular PCV2 (mut) were identified by indirect immunofluorescence assay (IFA).A pair of specific primers was designed to amplify the sequence of ORF1 gene(945bp) of porcine circovirus type 2 from lymph node of piglets with PMWS, and thisamplified production was cloned into pGEX-4T-1 expression vector. Then therecombinant plasmid (pGEX-4T-1-ORF1) was conformed by DNA sequencing andtransformed into E.coli BL21 strain. The fusion protein was expressed under theinduction of IPTG. The result of SDS-PAGE and Western blot analysis indicated PCV2ORF1 coding production was expressed successfully in E.coli BL21 strain, and theproduction has the most amount when expression was induced by 1mM IPTG for fivehours. This fusion protein can be identified by GST polyclonal antibody and itsmolecular weight is 63KDa.