Identification Of A Conservative Linear B Cell Epitope On The VP1Protein Of Duck Hepatitis A Virus Type1

Duck viral hepatitis is a kind of highly contagious and lethal infectious disease which caused by duck hepatitis virus. Duck hepatitis A virus type1（DHAV-1） can infect young duck mainly within the3-week-old ducklings. DHAV-1was first described on Long Island in1949（Levine and Fabricant,1950）. In1963, the disease was founded in the mainland of China, although the virus was not identified until1984. Duck viral hepatitis is caused by three different serotypes. Now DHAV-1is becoming a worldwide in distribution and one of the most economic importance disease. To date, there were not such more reports about the research of protein functions of DHAV. In this study, on the basic of obtained the whole genomome sequence and monoclonal antibodies for the DHAV-1, structural protein VP1of DHAV-1LY0801strain was cloned and expressed in the eukaryotic expression system for epitope research. This study includes the following:1. Preliminary analysis of the epitope to5specific monoclonal antibodies of DHAV-1In this study, five specific monoclonal antibodies against DHAV-1were prepared by intraperitoneal injection of mice. DHAV-1LY0801strains cDNA as the template, DHAV-1complete VP1gene was amplified by PCR, cloned into the baculovirus expression vector and transfected into sf9insect cells. By IFA, the epiopes recognized by the five monoclonal antibodies were located in the VP1protein DHAV-1. Western blot results showed that:tw7o monoclonal antibodies （4F8and5G4） recognized the linear epitopes, while the three others （1E10,4E6and5E11） recognized conformational epitopes.2. Identify of the accurate position of the linear epitope to monoclonal antibody4F8In this study, thirteen pairs of primers were designed and synthesized according to the genome sequence of DHAV-1LY0801stain available in GenBank. The sequences of DHAV-1LY0801stain were amplified by polymerase chain reaction （PCR） used the13pairs of primers. The products of PCR were cloned into pET-32a（+） vector, then the prokaryotic expression recombinant plasmid was transformed into Rosetta（DE3） PLys. The fusion proteins expressed in E. coli induced by IPTG, and the protein could react with positive serum by western blot. The antigenities of recombinant proteins were analyzed by SDS-PAGE, western blot with McAb4F8. The Bac-to-Bac Baculovirus expression recombinant plasmids with VP1fragments were constructed, and then transfected into sf9cells using collection reagent to obtain the product of recombinant viral stock. The antigenities of the recombinant proteins were analyzed by IFA. According to Western blot and IFA analysis results, combinated of the primer design, the epitope recognized by McAb4F8was speculated. The results indicated that the epitope recognized by McAb4F8was a linear epitope on the location of DHAV-1VP1protein74-82, corresponding to amino acids⁷⁴SGEIILTTV⁸², which was well conserved among DHAV-1. While this linear epitope doesn’t exist in DHAV-2and DHAV-3, which indicted that the McAb4F8could be used for immunodiagnosis of DHAV-1.