| As a member of the family Circoviridae, Porcine Circovirus(PCV) is a small, nonenveloped, icosahedral mammalian virus containing a circular single-stranded DNA genome. PCV type2(PCV2) is closed associated with Postweaning multisystemic wasting syndrome(PMWS). PCV2 also been associated to a number of pathological conditions of pigs, including porcine dermatitis and nephropathy syndrome,reproductive failure,porcine respiratory disease complex,proliferative and necrotising pneumonia and congential tremor. The wide preralence and spreading of the disease posed serious threat to pig industry and huge loss in many nations and regions in the world. The more serious thing was that the infection of PCV2 can depress the pigs immune system and lead to the second infection of other pathogens. Then, the more huge loss be made. Now the infection come from PCV2 has become one of the pathogens that can handicap progress of pig industry seriously.The paper was divided into 2 parts:1, Cloning and expression of ORF1 and ORF2 genes of porcine circovirus type 2. ORF1 and ORF2 genes of porcine circovirus type 2 were obtained by PCR amplify with the specific primers designed and synthesized according to PCV2sequence of GenBank. Following extraction and digestion, PCR products were inserted into pET-22b to generate two recombinant plasmids, pET-22b/ORF1 and pET-22b/ORF2. The recombinant plasmids were transformed them into E..coli BL21 (DE3) and expressed with IPTG induction. The expression products were analyzed by SDS-PAGE. Recombinant protein were mainly expressed as inclusion bodies in the system, and purified by Ni2+-affinity chromatographic column. The purified protein was refolded following dialysis. The result of ELISA showed the expressed products could react with the positive serum of pcv 2 specifically.2, Establishment and preliminary application of TaqMan fluorescence quantitative polymerase chain reaction method for detection of PCV2. The probes and primers were designed and synthesized according to the conserved gene PCV2available in GenBank, and then reaction parameters were optimized to develop a real-time TaqMan fluorescence quantitative PCR assay. The tissue samples from pigs were detected by using the established quantitative PCR assay, and the results was compared with that of routine PCR. The results indicated that the developed quantitative PCR assay could detect 3.0×101 copy /μL of plasmid DNA and its sensitivity was 10 times higher than that of the routine PCR.,and have no cross reaction with PCV1, CSFV, PRRSVand PRV. The real-time TaqMan fluorescence quantitative PCR assay which is specific, sensitive and accurate can be used for the diagnosis of PCV2 infection. |
