Cloning And Expression Of ORF1,ORF2 Gene Of Porcine Circovirus Type 2

Porcine circovirus type 2(PCV2) is a novel virus of the Circoviridae family was considered the cause of Postweaning piglets Multisystemic Wasting Syndrome (PMWS) which clinically characterized by wasting and death of postweaning pigs.PCV2 also been associated to a number of pathological conditions of pigs, including porcine dermatitis and nephropathy syndrome ,reproductive failure,porcine respiratory disease complex,proliferative and necrotising pneumonia and congential tremor type A II. The wide preralence and spreading of the disease posed serious threat to pig industry and huge loss in many nations and regions in the world. The more serious thing was that the infection of PCV2 can depress the pig,s immune system and lead to the second infection of other pathogens. Then, the more huge loss be made. Now the infection come from PCV2 has become one of the pathogens that can handicap progress of pig industry seriously. So far, there was not any good ways to control the infection of PCV2. The purpose of this experiment was to develop good diagnostics reagent and genetic engineering vaccine by Biomolecule method. The paper was divided into 2 parts. 1. The expression of ORF2 gene of type 2 Porcine Circovirus in E.coli; 2. Construction and expression of recombinant adenovirus carrying ORF1 gene of porcine circovirus type 2. 1.In order to express ORF2 gene of porcine circovirus type 2 (PCV-2),a pair of primers was designed according to the sequence of GD strain.The ORF2 gene was amplified use the primers that have Hind III and Kpn I site by PCR, and then the gene was subcloned into prokaryotic expressing vector pBAD/gIII.So the recombinant plasmid named pBAD/gâ…¢-ORF2 was constructed.After that,pBAD/gIII-ORF2 was transformed into Escherichia coli Top10 and induced by L-Arabinose. The results of SDS-PAGE and Western-blot indicated that the ORF2 gene was expressed and the recombinant fusion protein was about 28 Ku. 2.According to the sequence of PCV2 ORF1 gene,one pairs of PCR primers were designed and ORF1 gene were amplified from PK15 cells inoculated with PCV2 samples . The PCR products were cloned into pMD18-T vector and the recombinant plasmids were obtained.Then,the ORF1 gene was subcloned into the shuttle plasmid of pAdTrack-CMV.The recombinant plasmid and adenovirus backbone DNA were cotransformated into E.coli BJ5183 and the recombinant adenovirus genomic DNA was obtained. After transfecting 293 cell line with recombinant adenovirus the genomic DNA with the lipofectamine method,the recombinant adenovirus carrying ORF1 gene was screened under fluorescent microscopy, PCR analysis and Western blot analysis. The results demonstrated that the protein expressed by the recombinant adenovirus system could react with polyclonal antibody against PCV2 by Western blot, showly the some antigencity. This experiment use the expression system of coliform and the expression system of adenovirus to get the expression production of ORF1 and ORF2 gene of PCV2.The result can made very stability groundwork for diagnosis reagent and gene engineering vaccine.