Expression Of The VP0 Gene Of Duck Hepatitis Virus Type 1 And Development Of ELISA Based On Recombinant Protein

Duck hepatitis virus is a kind of virus without envelope, its structural proteins are closely related to reproduction of the virus and interaction between virus and host. Studies showed that high variation in both ends of VP1 protein of DHV-1(Duck hepatitis virus tpye 1) is not only the basis to differentiate from other picornavirus, but also contributes to the variation of different virulence of VP1 protein. However, structural protein VPO is relatively conservative. Based on the research of the HPeV(Human Parechovirus), which has closest relationship with DHV-1, it suggests that conservative B cell epitope may also exist on the VPO protein of DHV-1, which could induce the production of neutralizing antibody. Then the ELISA based on VPO protein can identify a wide range of serum antibodies against mainly strains of DHV-1. So it has broad application prospect for developing and commercializing ELISA detection kit.VPO protein of DHV-1 was expressed by employing molecular biological method, and indirect ELISA to detect serum antibody against DHV-1 was developed.They are three parts in this thesis.In the first part, VPO gene was cloned into pGEX-KG vector correctly. The recombinant expression plasmid pGEX-KG-VPO was expressed in E.coli expression system. The fusion protein VPO was expressed in E.coliBL21(DE3) and confirmed by SDS-PAGE. It showed that VPO fusion protein was expressed as two forms, soluble protein and inclusion body, both are purified and collected, which would be used in the next part.In the second part, soluble VPO-ELISA and inclusion VPO-ELISA were established based on the recombinant protein. Using the purified recombinant protein as coating antigen, the optimal conditions including the coating concentration and condition of the protein, serum sample dilution, HRP-rabbit-anti-duck IgY dilution and incubation time, were determined for the both ELISA methods. Both soluble and inclusion VPO protein showed no reaction with antiserum to the avian influenza, duck enteritis virus, duck reovirus and riemerella anatipestifer. It was shown that both ELISA were specific.Meanwhile, parallel comparison had been made between ELISA method based on the recombinant protein and ELISA based on DHV-1, and 978 serum samples were detected. Kappa value of soluble VPO-ELISA and virus-ELISA was 0.898, and the value of inclusion VPO-ELISA and virus-ELISA was 0.763. Studies have showed that two methods enjoy high consistency when the kappa value is above 0.6. So indirect ELISA based on recombinant protein VPO is considerably credible.In the third part, serum samples associated with DHV-1 infection were detected in some regions of Hubei and Henan provinces. For 114 serum samples in Hubei province, eighty-seven percent of which was detected positive by virus-ELISA, while ninety percent of these samples was positive by soluble VPO-ELISA. For 60 serum samples in Henan province, ninety percent of which was detected positive by virus-ELISA, while eighty-eight percent of these samples was positive by soluble VPO-ELISA.