Isolation And Identification Of Porcine Circovirus Type 2 Isolated In Shandong Province & Cloning And Expressing Of ORF2 Gene

Porcine circovirus is a member of Circoviridae, which can be divided into two types, PCV1 and PCV2. PCV2 can cause a disease named postweaning multisystemic wasting syndrome (PMWS), whereas PCV1 is ubiquitous and nonpathogenic for pigs. Since 1991, Canada has reported for the first time, PMWS rapidly in many endemic countries, there were also many PMWS infection and reports of virus isolation in China. PMWS has made a great loss in world's swine industry and aroused people's attention. In addition to PMWS caused by PCV2, with porcine dermatitis and nephropathy syndrome (PDNS), Piglet congenital brain tremor (CT) and other related, PCV2 infection of pig-raising industry has been seriously affected. According to the report of the clinical and laboratory tests, Shandong Province also has a certain amount of PCV2 infection, and actually had a severe effect on production. In order to further understanding of PCV2 and provide a scientific basis in the prevalence of Shandong Province, and further establish its rapid diagnosis of infection and quarantine methods for large-scale pig farms in the PCV2 diagnosis, prevention and treatment, we have carried out research.1.Isolation and Identification of Porcine Circovirus Type 2 Isolated in Shandong Province and sequence analysis of its ORF2 GeneAccording to the published gene sequence of porcine circovirus (PCV) from Genbank, a pair of primer specific to porcine circovirus type 2(PCV2) were designed and synthesized. Lymph nodes and spleen of a swine suspected PMWS in some porcine farm in Shandong province were detected by polymerase chain reaction (PCR). The result showed that 2 PCV2 positive reactions were detected in 8 samples. The positive rate is 25%. Which approved PMWS existed Shandong province. The PCV2 positive samples were abraded,sedimented by centrifugation,filtered and inoculated to the PK-15 cell line,which were not contaminated by PCVI. The extract viral DNA in PK-15 cells after 6 passages was detected with a pair of primers of PCV2 and obtained positive result. The result showed that the PK-15 cells free of PCV were infected by PCV2. Then the viral samples were pured and observed with electronic microscope. They were 17nm in diameter. In the cytoplasm,the specific fluorescence particles were observed in the frozen section strained by indirect immunofluorescence test specific to PCV2. According to above results,the isolated Virus strains belonged to PVC2 and named PCV2 SD 01 and SD02.According to the published ORF2 gene sequence of PCV2, a pair of primer were designed synthesized and ORF2 gene was amplified by PCR. The product was about 0.7 kb in size detected by agarose gel electrophoresis. It was In accord with the anticipated fragment. The products were purified and sequenced. The result showed that the ORF2 genes of SD01 and SD02 isolated strains were 702bp in size and they shared 99.9% homology with the referential strains and 91.9-99.4% homology with other isolated strains.2. Expression of ORF2 Gene of Porcine Circovirus Type 2 Isolated in Shandong ProvinceUsing other pair of primers, the ORFs gene, a part of ORF2 gene, was amplified by PCR. A fragment of 585bp of ORFs gene was obtained and cloned into pMD-18T vector and then transformed into expression vector pET-32a. After identification of the posivtive pET-ORFs with inserted PCV2 ORFs gene by by PCR,double digestion and sequencing, the recombinant were transformed into BL21 competent analysis cell of E.coli and induced by IPTG with a finial concentration of l mmol/L. SDS-PAGE showed that the recombinant protein had been expressed. The protein could react with polyclonal antibody against PCV2 by Westem-Blot test. The result declared that the expressing protein shared a good antigencity.Isolation and identification of PCV2 and sequence analysis of its ORF2 gene will supply good foundation for a etiology,diagnosis and control of PCV2. And the result also will be helpful to research the bioactivity of ORF2 protein-code and establish the diagnostic kit. The research about the expression of ORF2 Gene of Porcine Circovirus Type 2 has greater significance in disease diagnosis and vaccine research.