Establishment Of Indirect Elisa And Cleia For Detecting The Antibody Of Duck Hepatitis A Virus Type 1

Duck viral hepatitis(DVH) is an infectious disease of high mortality caused by duck hepatitis virus(DHV),which primarily infected ducklings with 21 days old. DHV leads typical hepatitis symptoms in duck with mortality rate of over 80%, and causes great losses to the poultry industry.. Virus isolation, virus neutralization test and viral nucleic acid detection were mainly used to the detection and diagnosis of DHV-1.Although they have their ownadvantages, they were not applied to the large quantities of clinical samples testing due to time-consuming. Accordingly, structural protein VP1 of DHAV-1 epidemic strains was cloned and expressed in E.coli Rosetta(DE3).Development of an indirect enzyme-linked immunosorbent assay(ELISA) and Chemiluminescent enzyme immunoassay(CLEIA)for detection antibodies to Duck hepatitis A virus type 1 using the purified recombinant VPlprotein as testing antigen.1. Cloning and Expression of VP1 gene for duck hepatitis A virus 1The VP1 gene fragment of DHAV-1 with 714 bp in length was amplified by RT-PCR, using RNA as the template. Recombinant expression plasmid pET-28a-VP1 and pET-32a-VP1were constructed to explore expression conditions of VP1 gene. They were transformed into E.coli Rosetta (DE3), inducing expression recombinant protein with different induction time and IPTG concentration. SDS-PAGE analysis revealed that:VP1 fusion protein with an approximate molecular mass of 27kDa and 47kDa, were expressed successfully by pET-28a-VP1 and pET-32a-VP1 respectively. In this study, pET-32a-VP1was used to efficiently express VP1 fusion protein, which could be purified by His-Ni+ affinity chromatography. Meanwhile, SDS-PAGE showed that VP1 fusion protein can be used as an testing antigen, on account of more than 95% purify.2. Development of an indirect ELISA and CLEIA antibody detection method for DHAV-1The intra-plat and inter-plate duplicability results of CLEIA are 2.27%-2.55% and 2.18%-4.33% respectively, less than the indirect ELISA of 1.61%-4.80%and 2.29%-8.60% respectively, indicating that the latter have higher stability, and CLEIA is suporior to indirect ELISA, providing a new means to diagnosis and monitor of DHAV-1.