Man B Lymphocyte Stimulator Factor Humanized Antibody Expression In Pichia Pastoris And The Development Of The Elisa Kit

B cell activating factor belonging to the TNF family (BAFF) is the seventeenth member of the tumor necrosis factor (TNF) ligand superfamily. It is a type II transmembrane protein, which can be expressed as a suface-membrane bound molecule or be proteolytically cleaved into a solube cytokine (134-285 amino acids, sBAFF ). The membrance-anchored form and soluble form of BAFF have similar functions. BAFF, under physiologic conditions, circulates in trimeric form and can regulate can regulate proliferation and function of T and B lymphocytes by binding to three different receptors, BAFF -R, TACI and BCMA. Lack of BAFF can lead to immunodeficiency, whereas overexpression of BAFF may result in B cell-mediated autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SS) and multiple sclerosis (MS) and serum BAFF levels correlate with its associated disease severity. Therefore BAFF A may be an attractive therapeutic target for human BAFF -associated autoimmune diseases.The gene, scFv-Fc, coding for the antibody of BAFF was inserted into the eukaryotic expression vector, pPICZαA, and transformed into Pichia pastoris. A high-level expression strain was obtained using a 'yeastern blotting' method. The scFv-Fc antibody was purified and 56 mg was obtained from 1L of culture supernatant. It proved that the antibody retained high binding activity to human BAFF by ELISA, western blotting and flow cytometry experiments.The hybridoma cells, which secrete anti-hsBAFF monoclonal antibody, were vaccinated into Bal b/c mice and the ascites was collected; the antigen was prepared to immune rabbits and the serum was collected. The monoclonal antibody and polyclonal antibody were respectively purified from ascites and serum by affinity chromatography. ELISA kit was established by coating with the monoclonal antibody and detecting with the biotin-labeled polyclonal antibody. The detection range of the kit was 1-100 ng/ml and it can be used in both research and diagnosis.