中国生物工程杂志

The gene of mutaed TNF-αD4 gene was amplified by overlap PCR and cloned into the prokaryotic expressive vector pBV220. TNF-αD4 contains two changes: substitutions of pro8arg,ser9lys,asp10arg,ile157phe,leu 29ser,arg31val and a deletion of the N terminal four amino acids . The recombinant vector pBV220-tnf-αD4 was transformated into E.coli strain DH5α, and the high expression strain was obtained by screening monoclones. The level of expression was about 45% of total cell protein. After purification, the purity of fusion protein was above 90% by HPLC and relactive ability was 8 ×107 .TNF-αD4 was modificated by mPEG-ButyrALD。After purification， the purity of mPEG-TNF-αD4 was above 85% and relactive ability was 8.6 ×107. and the in vivo systemic toxicity of mPEG-TNF-αD4, which is indicated by LD50, is lower than that of rhTNF-a .These results strongly supported for the further study and exploitation of TNF-antitumor drug.