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Human B lymphocyte stimulator humanized antibody expression in Pichia pastoris and ELISA kit developed

Author: RenFang
Tutor: ZhangShuangQuan
School: Nanjing Normal University
Course: Biochemistry and Molecular Biology
Keywords: B lymphocyte stimulator Heterologous expression Pichia pastoris scFv-Fc ELISA Kit Humanized antibody
CLC: R392
Type: Master's thesis
Year: 2008
Downloads: 125
Quote: 0
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Abstract


B lymphocyte stimulator (B cell activating factor belonging Io the TNF famlly, BAFF) is the seventeenth member of the tumor necrosis factor (TNF) superfamily , membrane-bound and soluble two forms . BAFF not only involved in the regulation of B cell proliferation , differentiation and antibody production , but is also involved in T-cell activation and response process . BAFF deficiency can lead to immune dysfunction ; BAFF overexpression closely involved involve both B and T lymphocytes the hyperthyroidism systemic lupus erythematosus ( SLE ) , rheumatoid arthritis (RA) and Sjogren's syndrome (SS) kinds of the occurrence of autoimmune diseases and Mao Fair , and the level of BAFF in serum is associated with the severity of the disease ; can alleviate symptoms of disease associated with anti - BAFF antibodies or soluble receptors inhibit the function of BAFF . Thus , BAFF has been considered to be an ideal target for the treatment of these autoimmune diseases . The first part : hBAFF Holistic derived genetically engineered antibody (scFv-Fc) in Pichia pastoris expression, purification and activity analysis of the study by recombinant DNA technology to build the B-lymphocyte stimulator . (BAFF) antibody genes ( scFv- the Fc) eukaryotic expression vector pPICZ ~ tA -scFv -Fc expression vector , and this electric into Pichia pastoris KM71H . \Affinity chromatography purified antibody per liter of bacterial suspension to give about 50 mg of purified antibody . ELISA, western blowing and flow cytometry assay , the antibodies can specifically identify hBAFF. The second part : hBAFF ELISA kits developed this study secreting anti hsBAFF monoclonal antibody hybridoma inoculated intraperitoneally into mice , the ascites was collected ; preparation of the antigen- immune rabbit serum was collected . Affinity chromatography and ascites and serum were purified to obtain monoclonal and polyclonal antibodies . The monoclonal antibody as coating antibody and a polyclonal antibody as a detection antibody labeled with biotin , made available ELISA kit for the quantitative detection of hBAFF concentration . The detection range of the kit 1-100ng/ml. The reagent cartridge has a good specificity and accuracy, can be used in the laboratory for quantitative concentration and clinical detection of cytokines .

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