Isolation And Identification Of PCV-2and Prokaryotic Expression Of Cap Protein

Porcine circovirus type2is the smallest pathogenic animal viruses which can causeweaning multisystemic wasting syndrome (PMWS), in addition, it also cause reproductivedisorders, colitis, weaned piglets and fattening pigs and other respiratory diseases, these diseasescollectively known as porcine circovirus disease (PCVAD). it has now spread all over the worldin each countries and become one of the major threaten in pig cultivation industry.In this study, a number of viruses samples isolated from pig farms in Guangdong andinoculated into PK-15cells and subcultured, isolate total RNA from PK-15cells inoculated withthe virus and reverse transcripted by means of RT-PCR. By indirect immunofluorescencepreliminary identification, we determined that the strains is PCV-2, according to the PCV-2GenBank sequence (SEQ ID NO: JQ809462.1), specificity primers designed for amplificationgenes encoding the full-length structural proteins, PCR technology used to amplified ORF2gene, confirm that the isolated virus is PCV-2.Using molecular biology techniques, PCV-2ORF2gene of GD10-01strain saved in ourlaboratory was connected to the pMD-18T cloning vector, according to the restrictionendonuclease digestion, we get recombinant plasmid PCV2-Cap-T. After sequencing correctly,verified by sequence analysis is correct, the target gene inprot into the prokaryotic expressionvector pET-28a-c (+) and get recombinant plasmid pET-28a-Cap. The recombinant plasmidswere transformed into Rosseta cells,37℃, IPTG induction for expression. The recombinant E.coli cell lysates were analyzed by SDS-PAGE, results showed that the recombinant bacteria cancorrectly express pET-28a-Cap and the protein has been purified already.Purified Cap protein used as an antigen to immune rabbit and get the polyclonal antibodies,simultaneously, the preliminary determination serologic titers was detected. An indirectimmunofluorescence assay was used to detect cells of porcine circovirus2. The optimumworking conditions has been established, the final fixed with70%acetone for10minutes in a1:200dilution of anti-1:400, using fluorescent secondary antibody reaction time60minutes.Indirect immunofluorescence method for detection of PCV-2provides the basis.