Isolation And Identification Of Henan Strain Of Porcine Circovirus 2 And Cloning, Sequence Analycis And Expression Of The ORF2 Gene

The epidemiclogical disease associated with PCV2 infection was investigated in 36 intensive swine farms in Henan from 2005 to 2007,the 1595 sera samples of PCV2 antibodies from 10 regions were detected for antibody against PCV2 by ELISA in two years.According to the published PCV2 gene sequences,one pair of special primer was desigened,and by using PCR,the 166 pathogency tissues were identificated from suspectedly infected swine farms.The results showed that PMWS caused by PCV2 occurred in 49 swine farms,PDNS and PRDC were present individually in 9 and 6 swine farms;The serum test results indicted that the total positive rates were 24.1%and 63.1% respectively.78%of tissue samples in 2005 and 96%of tissue samples in 2006 were positive for PCV2.It was indicted that the infection of PCV2 existed widely in swine farms in Henan,and more and more widely each year.Three primer pairs were designed in order to amplify the special genes of PCV1 and PCV2 and constructed multi-PCR.Two strains of PK-15 cells and some suspected infected PCV samples were detected.One strain PK-15 cells and three strains were only amplified PCV2 special gene.In the end,three strains of PCV2 were successful cultured.One pair of primers was desigened according to the published ORF2 gene sequence of PCV2. The PCV2 positive tissues by PeR with one pair primers were added to the PK-15 cells without PEV. Three strain of pcv2 were isolated from field samples by the PK-15 cell culture and were named KF,JZ and ZMD strain,The ORF2 gene was amplified from PK-15 cell strain infected with PCV2 by PCR..Then the PCR product was cloned into PMD18-T easy vector,and recombinant plasmid named PMD18-ORF2 yeas obtained.Analysis of ORF2 has shown that it was cloned successfully.Sequence and phylogenetic analysis confirmed that the extent of genetic heterogeneity of PCV2 Henan strain.The sequence of cloned PCR product was analyzed and compared with other PCV2 isolate in GenBank.It was found that shared 92.4%-99.6%and 90.6%-97.9%homology at their nucleotide and amineacid level respectively.Whereas ZMD strain displayed the highest nucleotide with American strain,JZ and KF strain displayed the highest nucleotide with Europeen strain.But ZMD strain has the highest homology with BF strain in China.Phylogenelic tree showed that ZMD strain is genetically closer to the American strain and JZ,KF strain is genetically closer to Europen strain.It is concluded that they might share a commen ancestor.Though ORF2 gene is generally stable among different isolate PCV2 isolates different geographic regions vary in their genomic sequences.Hydrophilicty prediction of protein encoded by ORF2 gene evidenced that antigenic epitopes has the same result with Truong's.According to the ORF2 gene analysis,It was modified by PCR using another pair of primers,a fragment of 587bp of ORF2 gene was obtained and cloned into PET-32a expression vector.The right recombinant plasmid was screened out and identified by enzyme digestion electrophoresis.The special protein was induced by IPTG and expressed,which is about 40KD.It could react with polyclonal antibody PCV2 by western-Blot,sharing a good antigencity.The intention of study is to investigate the epidimiology of PCV2,Henan strain relationship of the PCV2 is more complicated by compared with other ORF2 gene,and the expressed protein will give a base for the disease diagnosis and gene accine.