一种新型人肿瘤坏死因子突变体在大肠杆菌中的高效表达  

Overexpression of a Novel Highly Active Tumor Necrosis Factor Mutant in E. coli

作　　者：高长寿[1] 毛申兰 俞璎[1] 陈常庆[1] 

机构地区：[1]中国科学院上海生物工程研究中心

出　　处：《生物化学与生物物理学报》1996年第1期49-55,共7页

摘　　要：采用多位点突变引物和ＰＣＲ方法对合成的人肿瘤坏死因子进行了突变，通过这一突变，人肿瘤坏死因子的第二位精氨酸的密码子ＣＧＴ被变为赖氨酸密码子ＡＡＡ，将突变后的基因插入表达载体ｐＳＢ－９２的ＰＬ启动子下游得到重组质粒ｐＳＢ－ＴＮＦ－Ｋ２，并在大肠杆菌中进行表达。突变体［Ｌｙｓ２］ｈＴＮＦ－α的表达产率达到菌体内总蛋白质的６０％以上，且为一可溶性蛋白质并易于纯化。同野生型ｈＴＮＦ－α相比，［Ｌｙｓ２］ｈＴＮＦ－α具有稍低的毒性，但对于某些人肿瘤细胞株表现出高于数十到数百倍的抑制活性．A synthetic gene of human tumor necrosis faetor alpha(hTNF-α)was mutated by polymerase chain reaetion uaing a multi-site mutated primer with the Arg-2codon (CGT) replaced by a lysine codon (AAA). The mutated gene was inserted into vector pSB-92 at the downstream of PL promoter to give a recombinant plasmid pBS-TNF-K2 and expressed in E. coli. Soluble [Lys2] hTNF-α mutein was obtained in high yield, accounting for more than 60% of the total cellular protein and easy to purify. Compared to the wild type hTNF-α, purified [Lys2] hTNF-α has slightly less toxicity but shows tens to hundreds times greater inhibitory activity to several human tumor cell lines.

关 键 词：肿瘤坏死因子 高效表达 突变 

分 类 号：R392－33[医药卫生—免疫学] R394.8[医药卫生—基础医学]