Construction And Identification The Eukaryotic Expression Vector Of Porcine Circovirus Type2 ORF2 Gene And Porcine Parvovirus VP2 Gene

Porcine Circovirus Type2 is the primary causative agent of Post-weaning multisys- temic wasting syndrome,Sow abortion and mortality syndrome,Porcine dermatitis and nephropathy syndrome,and so on.PCV-2 could produce lesion of immune function.Because the imm- une function work irregular and prostration, the body could be infect with so much bacteri- um and virus,causing a potential economical impact on the swine industry world- wide.Porcine Parvovirus is the main pathogeny of Sow abortion and mortality syndrome.It possible cause abortion , stillbirth and mummification.Today, PCV-2 and PPV coinfection all over the world,we must to be faced with the draconic challenge.The PCV-2 and PPV were treated and inoculated into PK-15 cell.The DNA encoding full-length PCV-2 and PPV were extracted using prolease-K. Two pairs of PCR primers were designed according to the sequence of the PCV-2 and PPV 7909 strain.The DNA encoding ORF2 and VP2 were cloned into pCDNA3.1(+) to construct three recombinant plasmids containing ORF2 gene,VP2 gene, and ORF2-VP2 gene, were called pCDNA3.1- ORF2,pCDNA3.1-VP2 and pCDNA3.1-ORF2-VP2. Three recombinant plasmids were further analyzed with restriction enzyme. The result is, objective fragments were inserted into the pCDNA3.1(+) correctly.We extracted so many recombinant plasmids by the alkaline lysis and polyethylene glycol precipitation method.Analyze concentration and purity coefficient by detecting the Optical Density of the recombinant plasmids. The consequence is that they were good and do not have protein pollution.Healthy mouse were immunized with three recombinant DNA after pre-disposal treatment with Procaine. Injected 0.2mg recombinant DNA by intr- amuscular, two weeks later IM again till three times. We collect the blood-serum through removaling their globes at secondary immunization and third time week-later. Then we established indireet ELISA method for detectting the assays, the result shows that the mouse produce the antibidies to PCV-2 and PPV through IM three recombinant plasmids. Compared the secondary immunization with third times, latter had the conspicuous higher antibody level.In vitro study, cells that were transfected with pCDNA3.1-ORF2,pCDNA3.1-VP2 and pCDNA3.1- ORF2-VP2 were harvested at 48h postinfection.We extract their genomic RNA to amplified the objective DNA of PCV-2 and PPV with the RT-PCR methods. Cells that were transfected with three recombinant plasmids were harvested at two weeks postinfection. Using the mouse anti-ORF2 and VP2 antibody, the proteins were detected by western-blotting.These results show that ORF2 and VP2 could transcription and translation in transfected PK-15 cells. It is considered that the viral protein of PCV-2 and PPV have the immunogenicity that could stimulatedue the bodies produce the antibody to counteract the disease cause of PCV-2 and PPV. Maybe we could use the expression polyvalent antigen in controlling disease through both humoral-mediated immunity and cell-mediated immunity in the future.