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- alpha] production. An optimized RT-PCR was employed for construction of hTNF- alpha 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- alpha expression was assessed by an ELISA method using a monoclonal anti hTNF- alpha antibody together with a bioassay utilizing L-929 line as sensitive cells