Expression And Purification Of Ⅰ-DHV Vp1and The Preparation Of Monoclonal Antibody

Duck viral hepatitis (DVH)caused by duck hepatitis virus(DHV)is a highly mortal disease and spreads rapidly in four-week-old ducklings.DHV-Iis prevalent in China, which lead large economic loss for duckery. Taking the genome sequence of DHV-I strain EF14 as template,the target gene VP1 was cloned by RT-PCR. The obtained recombinant plasmids were named pET30a-VP1.DHV-I antigen of high purity was made in this research.It can be successfully used to carry out indirect ELISA and to immunize BALB/c mice for producing monoclonal antibodies.After a series of fusion and detection, three monoclonal antibody lines against DHV-I were prepared , and they have been applied tentatively,and could help to set uprapid diagnostic method for DHV-I in the future.A pair of PCR primers with two enzyme digested sites (BamH I and Xho I) were designed according to the sequence of DHV-I reported by GenBank. The full length gene fragment of VP1 was amplified by PCR, cloned into the pMD18-T vector and sequenced. The gene fragment of VP1 was inserted into the pET30a vector in order to construct the pET30a-VP1 recombined vector. Recombined vector was confirmed by RT-PCR and restriction endonuclease digestion . The protein was exist as inclusion body. After highly being expressed in E.coli BL21, the protein was purified and detected by SDS-PAGE and Western Blot.The purified protein use as antigen to immunize BALB/c mice to establish an indirect ELISA screening method. By repeated experiments before cell fusion to determine the conditions that indirect ELISA positive hybridoma cells selected optimum, the positive serum and negative serum were used as contrast. Mice spleen cells were fused with myeloma cells, three positive hybridoma cells were screened by indirect ELISA.The positive hybridoma cell lines were subcloned three times with limited dilution, named 2D9, 2D10 and 5F7. The monoclonal antibodies (McAb) were produced in ascites of mice by injecting 5×106 hybridoma cells.The stability of McAb subclasses, ascites titer and specificity were identified, the results showed that the monoclonal antibody had good specificity.Above all, three hybridoma cell lines anti-DHV-I VP1 antibody were established in this study.It demonstrated that we master the skills about culturing cells in vitro,detecting and purifying antigen, cell fusion. This study lays a firm foudation for producing more McAb, developing confirmation test kit and the research about DHV- I.