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Preparation and Characterization of Monoclonal Antibody Against DV Envelope Pretein

Author: TaoPing
Tutor: HuangAiLong
School: Chongqing Medical University
Course: Internal Medicine
Keywords: Dengue Monoclonal antibody Bionomics E protein
CLC: R446.6
Type: Master's thesis
Year: 2008
Downloads: 92
Quote: 0
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Abstract


Objectives: Dengue（DV） disease is a life-threatening disease. And nowadays the inciDVce of Dengue fever（DF） is increasing in the world. But we have had no utility vaccinum and specific medicine yet. So rapid diagnosis of the Dengue viral infection is important and emergent.The E protein , envelope protein of DV, is an important antigen protein and intimate to immunoprotection of Dengue infection. In this study, we constructed a prokaryotic expression vector to express DV-E in E.coli and obtained purified E fusion protein. Then, the specific monoclonal antibodies （McAbs） against DV-E were prepared by hybridoma cell techniques, which served as a basement of the gold colloid test paper.Methods:PCR was deployed to amplify E gene segment. After double enzyme digestion, prokaryotic expression vector pET32-a and PCR products were conjuncted by T4DNA ligase and converted into E.coli JM109 to construct and biopan the recombinant plasmid which was transformated into E.coli -BL21 later. After be induted by IPGT, the fusion protein was migration in SDS-PAGE gel electrophoresis analysis and detectived by Western-blot. Then the fusion protein was purified by Ni2+-NTA-chelated agarose affinity chromatography method.Balb/c mice were immunized with E fusion protein and monoclonal antibodies against DV-E were prepared with hybridoma method. The positive hybridomas secreting anti-E McAb were screened by ELISA. The antibody activity and specificity were analyzed by indirect ELISA assay. The McAb immunoglobulin （Ig） subtype and the titer of the selected E McAb were determined. The specificity of monoclonal antibody was tested by Western blot analysis.Results:The target gene segment, just the same size as expected, was amplified by PCR. The recombinant plasmid was successfully constructed and named pET32-a-E. And through be successfully transformating and inducting, we got the fusion protein, which molecularweight is 70kD, just as expected. Western-blot result indicated that the inductive expressed fusion protein could specificly bind with anti-DV serum. Through the Ni2+-NTA-chelated agarose affinity chromatography, we obtained the fusion protein with a purity of 90%.We screening out one hybridoma, named 7C7, the Ig subtype of 7C7 McAb is IgG1 subclass. Balb/c mice predisposaled were inoculated with well-grown hybridomas intraperitoneally. The induced ascites were proved containing anti-DV-E monoclonal antibody and their titer reached about 10-6; Western blot result suggested that McAb could bind specially to the antigen.Conclusion:The E fusion protein was obtained by recombinant DNA techniques and prokaryotic expression methods, and the specific monoclonal antibodies against DV-E were prepared. The obtaind monoclonal antibodies with high biological activity belong to the subtypes IgG1.

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