Expression Of The Cap Genes Of Porcine Circovirus And Preparation Of Monoclonal Antibodies

As a member of the family Circoviridae, Porcine Circovirus(PCV) contains two types genome:PCV1 and PCV2.PCV1 has no pathogenicity , PCV type2(PCV2) is relation to many dieases and closed associated with Postweaning multisystemic wasting syndrome(PMWS), The clinical mainfeastations of PMWS include poor body condition with varying degrees of muscle wasting, dyspnea and enlarged lymphnodes and abortion of gravid sow. The diversity of the incidence and the death rate of the diease is bigness, and which has caused great econmy loss all over the world. Recently it was approved that the major structural protein can diagnose PCV2 from PCV1 by ELISA,therefore the study of PCV2 is very necessary.Two pairs of primers specific to PCV1-Cap and PCV2-Cap were designed and synthesized according to the published sequence of PCV1 and PCV2 genes. Following the genes of PCV1-Cap was amplificated from strain U49186 and PCV2-Cap was amplificated from strain DQ104422. The two fragments were subcloned respectively into clone vector pGEM-T Easy . After testified, the two aim fragments were subcloned respectively into expression vector pGEX-6p-1 and GST-fusion proteins with appropriate restriction sites. The resulting plasmid contained the PCV1-Cap gene was designed as pGEX- PCV1 and the plasmid contained the PCV2-Cap gene was designed as pGEX-PCV2. By SDS-PAGE analysis, it confirmed that BL21 E.coli which contained pGEX- PCV1 or pGEX-PCV2 was able to express large quantities of about a 48-ku fusion protein (GST-PCV1 or GST-PCV2)The 8 weeks old BALB/c mice was immunized by using the fusion protein GST-PCV2 for 3 times(8 weeks,10 weeks,12 weeks). The third day after the last immunity, the splenocytes of immunized mice were fused with SP2/0 myeloma cells by a routine method, and the interested antibody were detected by indirect-ELISA and Dot-ELISA mothods. The hybridoma cell strain M1 and G1 were obtained, and M1 can stably secret monoclonal antibody specific for PCV2, G1can stably secret monoclonal antibody specific for PCV. Further more, indirect ELISA, Dot-ELISA, IFA by using the monoclonal antibodys were performed to detect PCV, and made a basis for furtherly setting up a PCV2-testing and PCV-testing method which has high specificity and ensibility, more over can be operated easily and quickly.