Expression Of Duck Circovirus Cap Gene And Establishment Of Indirect ELISA To Detecte Antibody To Cap

Duck circoviros(DuCV), which damages the host’s lymphatic system, is onenumber of the Circovirus in Cireoviridae. It induces immunosuppression mainly, and thengives rise to secondary infection or multiple infection. DuCV has catched more and moreattention. Up to now, the DuCV has been found and describes in several duck species suchas Cairna moschata, Muscovy duck, and Pekin-ducks in America; Hungaru; Germany;Taiwan etc, and it’s also widespread in FuJian、ShanDong、GuangDong and ZheJiangprovinces in China based on investigation report of the epidemic disease.DuCV cap protein is the important immunogenic protein to DuCV and a mainstructural protein in virus. The diagnosis method was found based on immunogenic proteinof DuCV, in order to provide a measure to diagnose DuCV in large-scale duck farm ofAnhui area. The details are as follows:Firstly, in this experiment, we analyzed the conservative region of bacterialstrain-DuCV AQ0901’s cap gene, and designed a pair of primes, The aim gene was gainedusing these special primes deleted several rare bases in the N-terminal; then clonned intoPET-28a plasmid, and identified by EcoRⅠ/SalⅠand sequencing. We expressed tHisrecombinant dcap protein in E. coli, and purified it by the Ni-NTA Purification system ofinvitrogen. Then we immuned Pekin-ducks with purified recombinant dcap protein andprepared polyclonal antibody serum. During the expression, we optimized the experimentalconditions including IPTG concentration, induction time and others, as well as detected theconcentration, purity, immunocompetence of recombinant protein. The results showed thatthe recombinant protein can express efficiently in E.coli and exist as a inclusion body bythe detections of SDS-PAGE and Western blot. The concentration of His-dcap was1.285mg/ml purified by NI purification resin. The antibody titer of polyclonal serum can reachto1:128.Secondly, an indirect ELISA based on the recombinant protein was developed. We thepurified recombinant protein as coating antigen reparated the negative and positive sera,selected the optimal concentration, determined the optimal coating conditions, andoptimized a series of time conditions for the best experimental results at the same time.Inthe new specific ELISA method, the optimum envelope antigen concentration and serumdilutability was6.57μg/ml and1:80respectively; the optimum time for enveloping antigenwas over night in4℃, and primary and secondary antibody incubation, coloration were1h, 1h and15min respectively. In this present study, at last338sera collected form severalduck farm in Anhui province had been detected using this new method, and the positiverate waas26.9%. The results suggested that DuCV were widespread in AnHuiprovince.The biological statistical methods was used in the analysis and it showed that theDuCV infection rates of Pekin ducks is higher in some areas of Anhui Province of ducks in30-60days of age segment is higher than any other age segment and the DuCV infectionrates varied at different reigons.The results confirmed that this method is effective and could be used in clinicaldetection for vast samples. This experiment provides a basic method to develop the capantinbody ELISA detective kit, and is useful to DuCV diagnosis and DuCV vaccinresearch.