Preparation and Pegylation of TNF-? Derivative / 中国生物工程杂志

ABSTRACT

The gene of mutated TNF-?D4 gene was amplified by overlap PCR and cloned into the prokaryotic expressive vector pBV220.TNF-?D4 contains two changes:substitutions of Pro8Arg,Ser9Lys,Asp10Arg,Ile157Phe,Leu29Ser,Arg31Val and a deletion of the N terminal four amino acids.The recombinant vector pBV220-TNF-?D4 was transformated into E.coli strain DH5?,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 relative ability was 8 ?107.TNF-?D4 was modificated by mPEG-ButyrALD。After purification,the purity of mPEG-TNF-?D4 was above 85% and relative ability was 8.6?107.The in vivo systemic toxicity of mPEG-TNF-?D4,which is indicated by LD50,is lower than that of rhTNF-?.These results strongly supported for the further study and exploitation of TNF-antitumor drug.