The Isolation And Indentification Of Porcine Circovirus (PCV)and Cloning And Sequence Analysis Of ORF2 Genes Of Porcine Circovirus Type 2(PCV-2)

According to the whole published gene sequence of PCV, two primers were designed , and 243bp fragment was amplified from suspected PMWS and PDNA samples by PCR-restriction fragment length polymorphis (PCR-RLFP). The result showed that 7 PCV positive samples were found in 12 samples. The average positive rate is 58. 3%. We digested PCR products with NcoI. In the end, we detected two PCV-2 DNA samples and a PCV-I sample in 7 PCV positive samples. Two PCV-2 strains, named with PCV-2 strain SCH A and PCV-2 SCH B strain and one PCV-1 strain named PCV-1 strain SCH C , were isolated from three samples in Sichuan Swine farms. Then the PK-15 cells free of PCV were infected by strain SCH A sample and strain SCH B sample. We only" detected PCV-2 SCH A strain virions in PK-15 cell after 15 passages by electron micograph.In this way, we detected PCV-1 DNA in IBRS-2 cells, named with PCV-1 SCH D;and we did not detect PCV DNA in PK-15 cells.According to the whole published gene sequence of PCV-2, two primers were designed , and 0RF2 gene was amplified from PK-15 cells infected PCV-2 by PCR. Then the PCR product was cloned into PMD18-T easy vector, and recombinant plasmid named PMD18-0RF2 was obtained. Analysis of 0RF2 has shown that it is 702bp. It is a major ORF, which encode 233 amino acid.To determine the extent of genetic heterogeneity of PCV-2 strain SCH A 0RF2 . Sequence and phylogenetic analysis confirmed that two distinct genotypes of PCV 0RF2 existrnonpathogenic genotpe PCV-1 and pathogenic genotype PCV-2. The genomic sequences of PCV-2 strain SCH A 0RF2 is closely related with strain HuNan isolated from China ; strain SCH A has 96% nucleotide sequence and 96% amino acid identity with strain HuNan. The data from this study indicate that although the genome of PCV-2 ORF2 is generally stable among different isolates PCV-2 isolates from different geographic regions vary in their genomic sequences. This variationmay have important implications for PCV-2 diagnosis and research.In this study, we suceed in founding special PCR-RLFP to detect PCV-1 and PCV-2 in tissue and cells . we get recombination plasmid of 0RF2 of strain SCHA. It approved that PCV-1 and PCV-2 are ubiquitions throughout our country. These works will give some help on expression , epidemiology, diagnosis, recombonation accine, PCV-1 or PCV-2 DNA infected clone.