Cloning And Expression Of Major Open Reading Frames Of Type 2 Porcine Circovirus

Two species of porcine circoviruses(PCV) have been classified into Circoviridae .They are small nonenveloped DNA viruses containing a covalent and closed single-stranded circular genome(1759 and 1768 nucleotides).Recently the study of porcine circovirus type 2 (PCV2) in abroad usually aimed at its replication ..transmission and epidemiology, but the factors triggering clinical expression are poorly understood-Therefore the objective of this study want to discover the potential open reading frame(ORF) function .Porcine circovirus type 2 (PCV2) has 5 open reading frame which encoded protein molecular weight more than 5KDa by analyzing its sequence.There are two major proteins have been encoded,0ne of the proteins associates with PCV2's replication named Rep.The other one is only caspaid protein of PCV2 named Cap.In this study , we want to set up a quickly and accurately diagnostic method. Two pairs of primers have been synthesized and amplified intact Rep's and Cap's gene fragments by PCR .Two fragments and another fragment about 400bp at the C-terminal of Cap were subcloned into vectors of pPROHTb and pGEX-6p-l gained recombinant plasmid of pPRO-Rep,pPRO-Cap and pGEX-ACap. To compare the antigenic reactivity of three recombinant proteins pGEX-ACap showing much stronger reaction than others.The result suggestion recombinant truncate ORF2 protein might be functional aspotential diagnostic of PCV2.The recombinant ORF2 and truncate ORF2 proteins might be functional as potential subvaccine against PCV2 infection.In order to understand the expression of proteins encoded by these potential ORFs in the course of PCV2 replication,then study the function of these proteins. Four pairs of primers have been synthesized and amplified intact ORF3, ORF4, ORFS and ORF6 gene framents.These framents were subcloned into expression plasmid pGEX-6p-l gained recombinant plasmid pGEX-ORF3,pGEX-ORF4,pGEX-ORF5,pGEX-ORF6.Specific antibodies were raised by immumizing rabbits with the purified fused proteins of GST-ORF3,GST-ORF4,GST-ORF5.GST-ORF6. Antisera were detected by Western blotting with PCV2 (supercentrifuge) The specific band being detected suggested that ORFS gene encoded protein might be expressed in the course of PCV2 replication.This gene fragment is not neither in cap gene nor in rep gene,but located in the complementary strand of rep gene ,and it will be studied further more.