表达载体pET22b-FGFR1的构建及在大肠杆菌中表达  

The construction of expression vector pET22b-FGFR1 and expression in Escherichia coli

作　　者：董媛[1] 冯淳[1] 马新宇[1] 曹欢[1] 郑诗卉 王会岩[1] 

机构地区：[1]吉林医药学院检验学院,吉林吉林132013

出　　处：《沈阳药科大学学报》2016年第4期313-316,320,共5页Journal of Shenyang Pharmaceutical University

基　　金：国家自然科学基金资助项目(81273421);吉林省教育厅课题吉教科合字[2014]第361号;吉林省大学生创新创业训练计划(2015036)

摘　　要：目的构建和表达人成纤维细胞生长因子受体1(fibroblast growth factor receptor,FGFR1)胞外区的基因工程菌,并对其表达条件进行优化。方法以肝癌细胞Hep G2为模板,利用反转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)扩增FGFR1胞外区,与载体pET22b连接后转化至大肠杆菌BL21(DE3)中,用乳糖对其进行诱导,经SDS-PAGE电泳分析诱导剂的浓度、诱导时机、诱导时间及诱导的温度等对表达量的影响。结果成功扩增出FGFR1胞外区片段,长度为600 bp。经酶切和测序证实获得的FGFR1胞外区基因序列与预期一致,构建的载体pET22b-FGFR1成功表达FGFR1胞外区蛋白,蛋白表达量质量分数在20%以上,最优表达条件为:当工程菌的A600的值为0.8时,终质量浓度为2.0 g·L^(-1)的乳糖一次性添加,37℃诱导3 h蛋白表达量达到最大,Western blotting分析该表达产物可以和人FGFR1多克隆抗体特异性结合。结论成功构建表达人FGFR1胞外区蛋白的基因工程菌,乳糖可以诱导FGFR1胞外区蛋白表达,为后续研究提供了良好基础。Objective To construct a recombinant E. coli strain expressing human fibroblast growth factor receptor 1（ FGFR1）,and optimize its expression condition. Methods Human FGFR1 gene fragments were obtained by RT-PCR with a template from HepG2 cells. Then the amplified fragments of FGFR1 were inserted into the pET22 b vector and the recombinant vector was transformed into the expression strain BL21（DE3） competent cell for identification. Recombinant protein was detected by SDS-PAGE and western blotting after induced by lactose. Finally,the effects of inducer concentration,induction time,temperature and OD value on protein expression were investigated. Results The target products of RT-PCR amplified fragments was about 600 bp,which was consistent with the expected fragment length. The recombinant vector of pET22b-FGFR1 was constructed and expressed successfully. The relative expression rate of FGFR1 was 20%（ w）. Optimization expression conditions were as follows：growth density of the strain（ A600） was 0. 8,lactose concentration was 2. 0 g·L^-1,and inducing time was at 37 ℃ for 3 h. Conclusions An engineering E. coli strain expressing human FGFR1 protein is successfully established and can be induced by lactose.

关 键 词：成纤维细胞生长因子受体1胞外区 乳糖诱导 表达 

分 类 号：R589.1[医药卫生—内分泌]