Expression And Application Of Truncated VP0and YP1Gene Of DHAV

Duck Viral Hepatitis is a highly contagious disease with hepatitis and hemorrhagic in young ducks, caused by DHV which is belonged to the family Picornaviridae. As the major structural proteins of DHV, both of VPO and VP1have very good immunogenicity. Through truncating VPO and VP1gene and screening the advantage hydrophilic and antigenic region, polyclonal antibody is prepared and a quick detection method of DVH is established.1. The expression of VPO and VP1truncated geneThrough amplifying five sections sequences VP0F1-3and VP1F1-2of VPO and VP1gene with a template and splicing them, VPO and VP1truncated gene were made. The VPO and VP1truncated gene was cloned into the expression vector pET28a to build a pET28a-VP0/VP1truncated recombinant plasmid which is induced by IPTG at16℃overnight conditions. The purified proteins is analyzed by SDS-PAGE and western blot. The result is that the purification effect and immunogenicity of VPO and VP1truncated protein is good.2. The establishment of an indirect ELISA detection method of DVH antibodyAn indirect ELISA detection method of DVH antibody was established with a coating antigen of VPO purified truncated protein and a secondary antibody of HRP-labeled goat anti-chicken IgY. By optimizing various conditions, the specificity high sensitivity and good repeatability method was established which the best coating concentration of VPO truncated protein is2μg/mL, the best duck serum dilution is1:320and the best HRP-labeled rabbit anti-duck IgY dilution is1:4000.3. Preparation of polyclonal antibodies against VPO and VP1truncated proteinThe immunogen of VPO and VP1truncated recombinant protein immunized a rabbits weighing about2.5kg for four times. When the titer is high, the blood serum is collected and purified to prepare the polyclonal antibodies against VPO and VP1truncated protein. The titer of polyclonal antibodies is tested by indirect ELISA with a coating antigen of purified DHAV. The titer of polyclonal antibodies against VPO truncated protein is51200and the titer of polyclonal antibodies against VP1truncated protein is12800.4. The establishment of a sandwich ELISA detection method of DVH antigenA sandwich ELISA detection method of DVH antigen was established with a coating antibody of polyclonal antibodies against VPO truncated protein and a detection antibody of duck polyclonal antibody. By optimizing various conditions, the high specificity and good repeatability method was established which the best coating concentration of polyclonal antibodies against VPO truncated protein is1:16000, the best duck polyclonal antibodies serum dilution is1:200and the best HRP-labeled rabbit anti-duck IgY dilution is1:3000.In conclusion, through screening the advantage hydrophilic and antigenic region of VPO and VP1gene, advantage antigenic VPO and VP1truncated protein is successfully expressed and polyclonal antibodies against VPO and VP1truncated protein is prepared. The specificity and good repeatability ELISA which is a quick detection method of DVH is established with a very important implications for disease prevention and treatment.