人三叶因子3在毕氏酵母中的表达及生物活性分析  

Expression of Human Trefoil Factor 3 in Pichia pastoris and Its Biological Activity Analysis

作　　者：王艳茹[1] 任宏伟[1] 安琳[1] 方敏[1] 李令媛[1] 茹炳根[1] 

机构地区：[1]北京大学生命科学学院,蛋白质工程国家重点实验室,北京100871

出　　处：《生物化学与生物物理学报》2001年第6期653-658,共6页

摘　　要：利用PCR方法在人胎儿胎盘cDNA中扩增了人三叶因子 3基因 (hTFF3) ,插入到含有AOX1启动子和α 因子信号肽序列的表达载体 pPIC9K中 ,采用毕氏酵母表达系统对其进行了高效的分泌表达 ,并用G418筛选高拷贝整合转化子。2 %甲醇诱导酵母表达 48h后 ,上清经SDS PAGE和Western印迹证明重组蛋白质以双体的形式分泌表达 ,占总蛋白质的 45 % ,能被抗hTFF3抗体所识别。表达产物经S Sepharose、Q Sepharose离子交换及SephacrylS 10 0纯化后纯度达到 95 %以上。体内生物学活性研究证实hTFF3可有效的防止盐酸诱导的大鼠胃溃疡。The human TFF3 (trefoil factor 3) DNA fragment was amplified by polymerase chain reaction (PCR) from human fetal placenta cDNA. The gene was cloned into the Pichia pastoris expression vector pPIC9K containing AOX 1 promoter and α factor leader sequence. Multi copies insertion transformants were screened on G418 plates. After the induction by 2% methanol for 48 hours, the expression of dimeric hTFF3 came up to 45% of total proteins in medium, as identified by SDS PAGE and Western blot assay. The recombinant protein was further purified by S Sepharose, Q Sepharose ion exchange chromatography and Sephacryl S 100 gel filtration chromatography to the 95% purity, as shown by densitometric scanning. The N terminus and molecular weight of the recombinant hTFF3 was in good agreement with the native hTFF3. The recombinant protein was proved to have good biological activity of preventing rats from the gastric ulcer induced by hydrochloric acid.

关 键 词：人三叶因子3 毕氏酵母 发酵 表达 生物活性 胃肠道粘膜修复因子 

分 类 号：Q786[生物学—分子生物学] Q781