Expression Partial VP2 Gene Of Canine Parvovirus And Establishment Of Double-antibody Sandwich ELISA And Its Application

Canine Parvovirus belongs to parvovirus families, Parvovirus genera. Parvovirus is the minimum and the simplest DNA virus among discovered virus. VP2 is the main structural protein of CPV and constitutes most of the viral capsid, The expressed overall VP2 protein was insoluble. Insoluble protein accumulates in inclusion bodies. It is necessary for recombinant protein soluble study to improve the exogenous gene expression quantity. CPV genetic engineering vaccine important protective antigen Canine parvovirus disease is mainly affects puppies that 2-6months. The infection rate was 100%, the mortality rates was 10%-50%. It became hotspot for prevent CPV to establish low cost, high detection rate, specificity, rapid and convenient detection method now.Using bioinformatics method, we analysis VP2 gene from isolated CPV-2a hydrophobicity plot and antigenic index, the partial length of VP2 gene from CPV was amplified by PCR using two pair of specific primers designed, and the PCR product was cloned into vector pEASY-T1. The recombinant vector was digested with BamH I and Xho I and inserted into the prokaryotic expression vector pET-32a(+) to obtain the recombinant expressed vector pET-32a-306,pET-32a-363 and it was transferred into the E.coli strain BL21 (DE3) cells. The expression of proteins was induced by IPTG and were purified and analyzed by SDS-PAGE and identified by Western blotting and indirect ELISA. The size of insert fragments was 30kDa and 33kDa; it can be specificity reactive with CPV antiserum. Two truncated VP2 genes of Canine Parvovirus were expressed in prokaryotic expression system to obtain recombinant proteins which could be used to develop subunit vaccine and diagnostic reagent.We purified CPV with PEG20000 and immunized New Zealand rabbit to prepare rabbit anti-CPV antibody.At the same time we collected mice ascites, which was produced from McAb cells. The two antibodies were purified with AKTA purifier. The size was 50kDa and 25kDa by SDS-PAGE.A double-antibody sandwich ELISA was established. In the optimized ELISA, rabbit original antibody was diluted 800 times with 0.05M carbonate as the capture antibody; monoclonal antibody was diluted 2000 times as the detection antibody. The best coating is 37℃1h and 4℃12h; the best reaction time of antigen is 37℃90min; It is blocked in 37℃for 2 h by 10% bovine serum;goat anti-mouse antibody labeled by HRP is diluted 4000 times and 37℃30min for response time;37℃10 min for color appearance. Sandwich ELISA can be used to detection CPV. Compared with RB of USA ELISA kit, the coincidence rate was 95%. The establishment of sandwich ELISA method would be a simple, quick and reliable method for screening large numbers of fecal samples of dogs suspected of CPV infection.