Sequence Analyzing And Prokaryotic Expression Of Orf2 Gene In The Strain Of Porcine Circovirus Type 2 Isolated From Lanzhou

Porcine circovirus type 2 (PCV2) is the main causative agent of Post-weaning Multisystemic wasting Syndrome (PMWS) and the other related diseases. Immunodeficiency is a well-known consequence of PCV2 infection, PCV2 is a immunosuppressive virus and induce other agents indirect infection, it can bring a great loss to our pig industry. It is often been ignored because of its sub-clinical infection, so the study of the epidemiology and molecular biology of virus should be future investigated. ORF2 gene of PCV1 and PCV2 is the main region encoding the structural protein, which has nature immunity and conservative gene sequence, so it is often been used in diagosis.In this study, PCV1 and PCV2 infected at the same time and very fast, so a pair of primers which were specific to PCV were desigend, that were used to amplify the DNA extracted from a pig tissues samples suspected PMWS by PCR test. It is used to lesion cell, PCR, Molecular Biology Methods and so on , that we have done ORF2 of PCV. Extract viral DNA in PK-15 cell after 5 passages infected PCV as template, and design a pair of primers that is ORF2 of PCV1 and PCV2 in common amplify the ORF2 gene of PCV. We were successfully cloned into T-easy vector, it was comfirmed by PCR, sequence analysis and restrict enzyme identification. We found the sequence of ORF2 shared 99% homology with ORF2 of PCV2, and which shared 68% homology with ORF2 of PCV1.On the basis of some ORF2 of PCV2 in GenBank and LanZhou isolates, purpose sequence be reformed and optimum allocation. Purpose sequence preferable represented antigenicity of PCV2. According to the ORF2 gene nucleotide and amino acid sequence analysis: The N and C end have a lot of rare codes, which influence the expression, So the ORF2 gene was modified. Synthetic protein cloned into pET-32a expression vector. The recombinant ORF2-pET 32a plasmid was transformed into BL21(DE3) competent cell of E.coli and induced by IPTG. SDS-PAGE analysis showed that the recombinant protein expressed was 50KDa in size, purification and western-bloting analysis showed the expressed protein can react with polyclonal antibody against PCV2.