Anti-htnf-¦Á Monoclonal Antibody Preparation Of Its Antibody Cloning Research

Tumor necrosis factor-a (TNF-α) is a multifunctional cytokine secreted by macrophages, NK and T cells, and TNF signaling may either induce cellular activation, apoptosis, or necrosis. TNF-a was thought to be a potent anticancer agent due to its cytotoxicity against a number of tumor cell lines in vitro and tumor in vivo. TNF-a was also known as a strong endogenous mediator of inflammation, and under pathological circumstance, overexpression of TNF-a sustains inflammatory phenomena characteristics of some disorders, such as tissue damage, grave septic shock and autoimmunity disease. As a key cytokine in regulation of physiologic equilibrium, TNF-a plays a "double-edged sward" role on cancer and other diseases in clinic.Several approaches using differently engineered proteins to block TNF have been developed. At present TNF was the first cytokine to be fully validated as atherapeutic target for RA,tumors. A series of preclinical studies based TNF therapies have unequivocally demonstrated a significant benefit in RA patients.Expression vector of hTNF a was constructed by cloning hTNF a DNA fragment into the expression vector pAYZ. The fusion protein was soluble expressed in periplasmic space, which was purified by E-tag affinity chromatography. McAbs against hTNF-a were prepared by traditional hybridoma technique. ELISA and Western blot were used to identify their specificity and immunoglobulin subtype. Ascitic monoclonal antibody was precipitated by saturated ammonium sulfate and purified by Protein G affinity column. hTNFa-antagonistic activity of McAbs were evaluated by inhibition of hTNFa-mediated cytotoxicity and apoptosis on murine L929 cells using MTT and FACS assay. The hTNFα-antagonistic monoclonal antibody (XB10) was prepared, the subtype was lgG2. XB10 antibody showed higher inhibitory activity in hTNF-a-mediated (1.0ng/ml) cytotoxicity(1.14 ug/ml of IC50) and apoptosis on murine L929 cells with a dose-dependent fashion. Continuously, the heavy chain/light chain genes were cloned by RT-PCR from the hybridoma cell line(XB10), and according to the antibody genes library of PDB, the sequence reports were analyzed by computer-aid design, and the results showed that we succeeded in cloning the heavy chain/light chain genes of XB10.