Prokaryotic Expression Of VP1-/3D- Encoding Genes Of New Duck Hepatitis Type 1 Virus And Preparation Of Monoclonal Antibodies Against 3D Protein

Duck viral hepatitis(DVH), an epidemic disease caused by duck hepatitis virus(DHV), is known as an acute, highly lethal, fast spreading epidemic with 100% fatality rate, and cause serious harm to ducks industry. It was first reported in USA 1945, after that, most countries of the world followed. The first discovery of new type duck hepatitis virus (DHV-1C) was 1994, in South Korea. In this study, prokaryotic expression was preformed to get structure protein VP1 and nonstructure protein 3D of DHV-1C N. After Westen-blot to test the immune activity, mice was vaccinated with purified 3D protein as immunogen. After cell fusion, positive selection and hybridoma cell cloning, a strain of monoclonal antibody against 3D protein was prepared successfully and indirect-ELISA method for screening was established. This is the foundation for further research of function of VP1 and 3D and also the foundation for establishment of quick test of DHV-1C.The primers specific for VP1-/3D-encoding genes of DHV-1C N strain were designed by using Primer 5.0 software. BamHⅠand XholⅠwas introduced into the primers of VP1, BamHⅠand HindⅢwas introduced into the primers of 3D. PCR was preformed with cDNA of DHV-1C N strain as template and with the primers designed. To construct VP1 and 3D expression plasmids, the PCR products were cloned into expression vector pET-30a, named BpET-30a-VP1-3 and BpET-30a-3D-8 respectively. Recombined BpET-30a-VP1-3 and BpET-30a-3D-8 were transformed into BL21. The highest expression of VP1 and 3D were 3h and 4h, respectively. SDS-PAGE analysis showed that the VP1 and 3D products were 37.68 kDa and 65.80 kDa, respectively. Western blot demonstrated that VP1 and 3D proteins reacted specifically with duck anti DHV-1C serum. Both of them had specific immune responses to positive serum, the immunocompetence was well. The expression products of VP1 is about 37.68 kDa and 3D is 65.80 kDa. Soluble analysis showed that both of them existed in bacteria in the form of inclusion body.Purified 3D protein was vaccinated BALB/c female mice as immunogen by intrapetitoneal injection and hypodermic injection. At the same time, using purified 3D protein as coated antigen to establish indirect-ELISA method. The third day after strengthen immunization,splenocyte of vaccinated mice was fused with myeloma cell SP2/0. A strain of hybridoma cell secreting monoclonal antibody against 3D protein of DHV-1C was obtained by indirect-ELISA method established above and named 2D9. The subtype of 2D9 was IgG1 withκlight chain. Western-blot analysis and indirect-ELISA determination showed that specificity of 2D9 was quite well and there is no cross-reaction withσB protein of reovirus. Dot-ELISA analysis indicated that 2D9 can recognized the nature structure of 3D protein. Color reaction and immunofluorescence reaction between 2D9 and cell infected DHV-1C was observed by Immunohistochemistry and immunofluorescence assay. The measure of antibodytiter of supernatant of 2D9 cell was 1:512,of ascites was 1:256 000. The hybridoma can steadily secrete antibodies after revitalization after three months cryopreservation and culturing consecutively 30 generation.