Isolation And Identification Of Porcine Circoyirus Type2from Xinjiang And Construction Of Enfectious Clone

Porcine circovirus type2(PCV2) was the primary pathogen of postweaning multisystemic wasting syndrome (PMWS), This virus can invade the immune system of pigs, causing immunosuppression.Firstly, The nucleic acid fragyments of PCV2were detected from the tissues of clinical pigs with PMWS by PCR. The positive samples were homogenized in PBS (pH7.2). The suspension was inoculated in PCV1free PK-15cells. After5passages, one isolate of porcine circovirus type2was isolated. The virus antigene was dected through indected immuned flouence test (IFA) and PCR. The result primary showed it is a PCV2.Secondly, One pairs of primers were designed according to the published PCV2complete genome sequence in GenBank. The complete genome of isolate strain was cloned by PCR, The result of sequence analysis showed that the isolate’s complete genome was composed of1767nucleotides, compared with PCV1and PCV2reference strains from GenBank. It shared76.8%homology with PCV1and shared94.1%-99.3%homology with PCV2. The isolate strain was named XJ-105.Finally, Two pairs of primers were designed according to the published PCV2genome sequence in GenBank. The first pair of primer was used to amplify the complete genome by PCR from the cells which were infected by XJ-105. The PCR product was cloned into pMD Simple18-T vector to construct the recombinant plasmid designated as T-PCV2. Then regard it as a template, amplifying another of complete genome of PCV2by use of the second pair of primer and cloned it into anther vector pBluescript II SK(+) to construct the recombinan plasmid of K-PCV2. at last, ligated the complete genome of T-PCV2to the upsrem of K-2PCV2to construct double copies of cloned strain of PCV2. Then Transfect PK-15cells with K-2PCV2. After five passages, the infectivity of the cloned strain of PCV2was confirmed by RT-PCR and IFA. The result primary showed it is a infectious cloned srtain of PCV2.In this study, one isolate of porcine circovirus type2was isolated from the tissues of clinical pigs with PMWS and it’s complete genome was amplified by PCR, the nuclectide sequences were determined. Phylogenetic tree analysis showed:there was correlation between PCV2isloates. Consequently, we constructed the double copies of infectious cloned of PCV2. All of these achievements will give some help on studying epidemiology, pathogenic mechanisms, replication mechanisms and vaccine development of PCV2.