人肿瘤坏死因子cDNA衍生物在大肠杆菌中的表达及活性测定

The Expression and Activity Assay of Human TNF Derivative cDNA in E. coli

应用基因工程方法构建了含人肿瘤坏死因子ＣＤＮＡ衍生物的重组体．转化大肠杆菌后酶切鉴定．转化菌经摇瓶培养及温度调控，获得高效表达．ＳＤＳ－ＰＡＧＥ结果表明，表达的ＴＮＦ分子量约为１７ｋＤ，蛋白量约为菌体可溶性蛋白的２０％．用Ｌ９２９细胞毒性测定法测得菌液ＴＮＦ的表达为２×１０８ｕ／ｍＬ．抗ＴＮＦ的单克隆抗体可以中和大肠杆菌表达的ＴＮＦ对Ｌ９２９细胞的细胞毒性．

A recombinant of human TNF derivative cDNA was constructed by genetic engineering. After transformed into E. colt,it was identified by restriction endonuclease analysis.Raising the incubation temperature from 28℃ to 42℃,a high leval expression of TNF was obtained. The result of SDS-PAGE showed that the nolecular weight of expressed TNF was 17 kDand The cytotoxity of expressed TNF to L929 cell line was 2 ×108 units in 1 mL cell culture and it could be neutralized with TNF monoclonal antibody.