Isolation, Characterization And Expression Of The Complementary DNA For Human Tumor Necrosis Factor (TNF)

Tumor necrosis factor (TNF) is a cytokine secreted by activated macrophages. A typical TNF activity has been associated with in vitro and in vivo specific killing of tumor cells, yet has no known effect on normal non-transformed cell lines. Mature human TNF is a non-glycosylated and moderately hydrophobic protein of 157 amino acids, corresponding to. a molecular weight of 17,000 Da.Besides tumoricidal activity, TNF is found to be an antiviral reagent, which can inhibit viral replication and pretect the cultured cells from viral infection. TNF can suppress lipoprotein lipase activity, enhance the proliferation of normal cells, stimulate bone resorption and inhibit bone formation. These results suggest that TNF may play an important role in the effects of endotoxin-mediated septic shock, in regulating the proliferation and differentiation of cells, and in control of normal bone remodeling. TNF has also been shown to modulate the immune system by enhancing the cytotoxic and phagocytic activity of polymorphonulcar neutrophils, activating macrophages and lymphocytes, inducing other cytokines (e.g. IL-1, M-CSF, β-IFN), increasing the expression of major histocompatibility complex antigens, and having the synergistic effect with IL-1, IL-2, and interferons. In addition, several phase I and II clinical trials using TNF in cancer patients have been started. TNF would be a potential biotherapeutic reagent in anti-tumor and anti-virus treatments, especially in anti-HIV (AIDS) practice.Nevertheless, the studies on the theory and clinical application of TNF has not been extensively carried out in our country for lack of the TNF product. The better way to solve this problem may be the production of tumor necrosis factor using recombinant DNA techniques.In the work presented here, the complementary DNA for human TNF was first isolated, cloned and expressed directly in Escherichia coli in our country. The strategy and results are as follows;1.The expression of TNF mRNA from human promyelocytic leukemia cell line HL-60 treated with PMA (phorbol myristate acetate).2.Isolation of poly(A)+RNA from induced IIL-60 cells and synthesis of cDNA by reverse transcription; then construction of a cDNA library containing 2.0×10~5 recombinants.3. A cDNA for (?)ing mature human TNF was (?) by (?) polymor(?)chain reaction (PCR) with 1st-stranded cDNA template and cloned in a plasmid.