Cloning and expression in Escherichia coli of the gene for mouse tumor necrosis factor

Abstract

The mouse tumor necrosis factor (TNF) gene was isolated from a mouse genomic library. The entire sequence of the gene was determined using both an automated DNA sequencer with improved primer extension reaction conditions as well as the standard radioisotopic method. Comparison of the nucleotide sequence of the gene with that of mouse TNF cDNA showed that the mouse gene consists of four exons, like rabbit and human TNF genes. There is strong nucleotide sequence homology in the 5'-flanking region among the mouse, rabbit, and human TNF genes, suggesting that the mechanisms regulating TNF gene expression are highly conserved. Direct expression of mature mouse TNF was achieved using a plasmid constructed by site-directed mutagenesis. Purified mouse TNF produced in Escherichia coli showed cytotoxicity to mouse L cells.