Expression and Purification of Ⅰ-DHV Vp1and the Preparation of Monoclonal Antibody
|School||Northeast Agricultural University|
|Course||Clinical Veterinary Medicine|
|Keywords||Duck hepatitis virus VP1 protein Expression and Purification Monoclonal antibodies|
Duck viral hepatitis (DVH) is the rapid spread of highly lethal infectious disease caused by the the duck hepatitis C virus (DHV) a major following 4-week-old ducklings. Duck hepatitis outbreak in China is mainly caused by the DHV-I, ducklings infected with DHV mortality was 100%. In this study, DHV-I strains EF14 VP1 gene was amplified by RT-PCR. Recombinant plasmid, named pET30a-VP1. And preparing a high purity DHV-I antigen, this immunization of Balb / c mice and to establish indirect ELISA screening method. After a series of fusion screening, successfully developed anti DHV type I monoclonal antibody 3. The experimental laboratory to establish DHV rapid diagnostic methods, and future research laid the foundation for the DHV-I. Whole genome sequence published under GenBank DHV-I design the synthesis of a pair of primers (containing BamH I and Xho I restriction sites), VP1 gene was amplified by PCR and cloned into pMD18-T vector and sequencing. Positive VP1 gene inserted into the expression vector pET30a to construct the recombinant expression plasmid pET30a-VP1, PCR and restriction analysis, and its high-level expression in E. coli BL21. The fusion protein form of inclusion bodies after sonication, exogenous gene expression using SDS-PAGE electrophoresis stripe size is in line with expectations. And the fusion protein was purified. Indirect ELISA screening methods as antigen to immunize Balb / c mice with purified protein. Cell fusion prior to trial to determine the optimal reaction conditions for the indirect ELISA screening positive hybridoma cells, positive serum and negative serum were used as positive and negative controls. Take the spleen cells of the immunized mice with myeloma cells SP2 / 0 fusion screened three positive hybridoma cells by an indirect ELISA method, the obtained positive cell lines were carried out three times by limited dilution subcloning were named 2D9, 2D10 and 5F7. Finally, the expansion of cultured hybridoma cells injected into Balb / c mice intraperitoneally to produce ascites, each injection of 2 × 106 cells, and preparing monoclonal antibodies. The stability of these three monoclonal antibodies, antibody subclasses, ascites titer and specificity of the identification, the results show that the monoclonal antibody obtained in this experiment are having a good specificity. The success of this study were prepared hybridoma cell lines secreting anti-DHV-I VP1 protein-specific antibody tests that have mastered the in vitro cell isolation and culture, of antigen identification purified cell fusion technology for fabricating three single resistant, developed antigen detection kit and further study of duck hepatitis virus type I laid the foundation.