Expression Of P1-ORF2 In Prokarvotic Cells And Preparation And Identification Of Its Monoclonal Antibodies

Porcine circovirus was divided into the family porcine circovirus,genus porcine circovirus,which includes two serotypes:PCV1 and PCV2.The main pathogen of postweaning multisystemic wasting syndrome (PMWS) is PCV2, which can induce the immunosuppression of the sicken livestock and cause secondary infection eventually.It is an important disease pathogen that causes heavy loss of pig industry.We got two strains of Procine Circovirus Type2-like (we call them Agent P1 and Agent P2 temporarily) from the serum of the PMWS pig. Agent P1 has 648 nucleotides and part of them have a lot of homology with PCV2. Although there is lots of progress on the research of the biological characteristics and differential diagnosis of the Agent P1,the structure and function of the protein that the genes code is still unclear.So the preaparation of its monoclona antibodies is very important for the next study of the Agent P11. Expression and identification of P1-ORF2 in prokarvotic cellsThe gene of ORF2 Agent 1 was amplified by PCR and inserted into prokaryotic expression vector pET32a.After been digested by the restriction endonuleases:BamH I and Xho I, the recombinant vector was transformed into E coliBL21(DE3) pLys. Then we got the fusion protein that its molecular weight is about 34,000 Dalton. The maximum expression product amount was obtained when the induction temperature is 20℃, the induction time is 12h, IPTG's concentration was 1.0mmol/L and the time of the bacteria's propagation before added IPTG was 0.5h.The analysis of Western-blot indicated that the fusion protein can react with the goat anti-rabbit hyper-immune serum of the Agent P1.2. Preparation and identification of the monoclonal antibodies against Agent P1-ORF2The analysis of sequence alignment shows that one base (A) is lost in the position 371 in the Agent P1's sequencce compare with the reverse sequence of the PCV2 which is in the position 461.Put this period of gene into two sections which is called:P1ORF2a and P1ORF2b. Amplify the two genes by PCR and inserted them into eukaryotic expression vector pcDNA3.0 seperately. Spleen cells from BALB/c mice single intrasplenic immunized with plasmid DNA were fused with SP2/0 myeloma cells after immunization. After three times of limiting dilution assay,four strains of hybridoma cell (A6,D10,H9,B8) were obtained,and their subtyps were all IgM.The ELISA titers of ascites were 102,103,103,105. The analysis of Western-blot and IHC indicated that the four strains of monoclonal antibody can react with the fusion protein of P1 ORF2 and not the PCV2 ORF2.