RT-PCR Cloning and Expression of Complementary DNA for Human Tumor Necrosis Factor Alpha

Abstract

U-937, a monocytic cell line was induced with Phorbol Myristate Acetate (PMA) for human tumor necrosis factor alpha (hTNF-α) production. An optimized RT-PCR was employed for construction of hTNF-α complementary DNA (cDNA). The resulted fragment was verified by restriction digestion mapping with PvuII.The verified fragment was cloned in pUC18 plasmid and transformants were pelletted onto LB agar medium containing ampicillin, X-gal and IPTG. The resulting white colonies were verified by PCR and cultured in LB medium containing ampicillin and IPTG. The biological activity and the quantity of hTNF-α expression was assessed by an ELISA method using a monoclonal anti hTNF-α antibody together with a bioassay utilizing L-929 line as sensitive cells.