融合表达载体pET22b-SUMO-FGFR4的构建及其在大肠杆菌中表达条件的优化  

Construction of fusion expression vector pET22b-SUMO-FGFR4 and optimization of expression conditions in E.coli

作　　者：刘微[1] 姚杨[1] 马萧萧[1] 邓裕宣 梅迪[1] 刘磊[1] 王会岩[1] 

机构地区：[1]吉林医药学院检验学院生物技术教研室,吉林吉林132013

出　　处：《吉林大学学报（医学版）》2016年第4期642-647,共6页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(81273421);吉林省卫计委青年项目资助课题(2015Q042);吉林省吉林市科技局科技发展计划项目资助课题(20156427);吉林省教育厅大学生创新创业训练项目资助课题(2015032)

摘　　要：目的:设计合成小泛素修饰物-成纤维细胞生长因子受体4(SUMO-FGFR4)基因,构建pET22bSUMO-FGFR4表达载体,并对其表达条件进行优化。方法:采用Overlap PCR方法制备SUMO-FGFR4融合基因,并连接到原核表达载体pET22b中,获得pET22b-SUMO-FGFR4重组表达载体。以乳糖为诱导剂,观察乳糖浓度、诱导时机、诱导温度、诱导时间和乳糖的添加方式等因素对SUMO-FGFR4蛋白表达量的影响,确定最佳诱导条件,并进行重组蛋白的可溶性分析。结果:pET22b-SUMO-FGFR4表达的融合蛋白在相对分子质量40 000处显示目标条带,并与FGFR4抗体特异性结合。融合蛋白在乳糖终浓度为1.0g·L-1、诱导时间为3h、诱导时机A(600)值为0.8、诱导温度为37℃时表达量最高,乳糖的添加方式对SUMO-FGFR4融合蛋白的表达量无明显影响。乳糖作为诱导剂比传统诱导剂IPTG诱导SUMO-FGFR4融合蛋白的表达量高7.5%,融合蛋白以包涵体形式为主。结论:以乳糖作为诱导剂,成功表达了SUMO-FGFR4融合蛋白,确定了融合蛋白的最佳表达条件。Objective：To design the small ubiquitin modification-fibroblast growth factor receptor 4（SUMOFGFR4）fusion gene and construct the expression vector pET22b-SUMO-FGFR4,to optimize the expression conditions.Methods：The SUMO-FGFR4 fusion gene was obtained by Overlap PCR and was connected to pET22b;the recombinant expression vector pET22b-SUMO-FGFR4 was obtained.The influence of lactose concentration,induction time,induction temperature,induction point and adding mode of lactose in the expression levels was observed,and the best induction condition was determined;then the solubility of recombinant protein was analyzed.Results：The SUMO-FGFR4 fusion protein was highly expressed,the molecular weight of the fusion protein was about 40 000 and it could bind with FGFR4 specific antibody.When the lactose concentration was1.0g·L-1,the induction time was 3h,the induction temperature was 37℃,the value of A（600）was 0.8,the expression level was highest;but adding mode of lactose had no remarkable effect on the protein expression.The expression level of recombinant protein induced by lactose was higher than IPTG.SUMO-FGFR4 protein existed in a form of inclusion body.Conclusion：The SUMO-FGFR4 fusion protein is expressed successfully in this study while lactose is used as inducer and the best expression conditions are confirmed.

关 键 词：小泛素相关修饰物 成纤维细胞生长因子受体4 重组融合蛋白类 

分 类 号：Q78[生物学—分子生物学] R73-3[医药卫生—肿瘤]