Construction of eukaryotic expression vector and establishment of cell line solely producing human TNF-αin secretory form
HUANG Jia qiang,BU Xia, SHI Xu, LI Zhuo ya, GONG Fei li Department of Immunology, Tongji Medical University, Wuhan 430030, Hubei Province, China
Aim To reveal the differences between membrame bound TNF αprecusor(mTNF α) and secretory TNF α(sTNF α)in the bioactivities. Methods A recombinant TNF with only secretory form(pBK sTNFm) was constructed in which the leader sequence of TNF αwas replaced by the DNA sequence coding the human IL 2 signal peptide and then transfected into NIH3T3 cells. Results G418 resistant colonies and their expression products were identified by bioassay and Western blot. TNF with Mr 17 000 could be demonstrated both in cell lysate and culture supernatants of the pBK sTNFm transfected cells and only the supernatant displayed cytotoxicity. Conclusion A cell line only producing sTNF αis successfully established, which also provides the basis for studying the biologic activity and its mechanism.