人肿瘤坏死因子-α突变体在大肠杆菌中的表达及活性测定  

Expression of a human TNF mutant in E.Coli and measurement of its bioactivity

作　　者：王潇 吴敏[1] 沈心亮[2] 应莲芳[2] 翟雷[2] 郑荣梁[3] 

机构地区：[1]兰州军区联勤部军事医学研究所 [2]卫生部兰州生物制品研究所 [3]兰州大学生物系

出　　处：《兰州医学院学报》1999年第1期7-9,共3页Journal of Lanzhou Medical College

摘　　要：目的构建一种新型人肿瘤坏死因子（ＴＮＦα）分子，测定其蛋白表达和生物学活性。方法在详细分析ＴＮＦα结构及其结构与功能关系的基础上，设计并人工合成了一对ＴＮＦα结构基因点突变引物。应用ＰＣＲ分子克隆技术构建了一种新型ＴＮＦα分子的编码基因，将该编码基因插入表达质粒，转化大肠杆菌，通过温度诱导获得了表达蛋白，对表达产物进行免疫学检测和生物活性检测。结果新构建的突变体ＴＮＦαΝ１与ＴＮＦαＥＬＩＳＡ试剂盒有免疫学反应，其含量为０．５μｇ／ｍｌ；在体外有生物学活性，为１０７ＢＵ／ｍｌ，比活为２．１×１０５ＢＵ／ｍｇ；理化性质与天然原型ＴＮＦα有所不同。结论得到了一种具有生物学活性的新型ＴＮＦα分子。Objective To synthesize a novel TNF and measure its bioactivity and expression. Method On the basis of analysis of TNF structure and the relationship between structure and function,a novel TNF coding sequence was synthesized by PCR technique and inserted into an expression plasmid.The novel TNF simultaneously made two alterations.Seven amino acids at amino terminus of TNF were deleted and ProSerAsp on 8910 sites were altered to ArgLysArg.Leu on 157 site was altered to Phe. Results By temperature induction,the transformed E.coli with the novel TNF expression plasmid produced high yield of novel TNF.Results showed that TNF protein content was 0.5 g/ml and the specific activity was 2.1105 BU/mg and the chemical and physical properties of novel TNF were different from those of nature TNF. Conclusions A novel TNF with bioactivity was constructed.

关 键 词：肿瘤坏死因子 PCR 基因突变 大肠杆菌 

分 类 号：R73－36[医药卫生—肿瘤]