吉林大学学报(医学版) ›› 2016, Vol. 42 ›› Issue (04): 642-647.doi: 10.13481/j.1671-587x.20160402

• 基础研究 • 上一篇    下一篇

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

刘微, 姚杨, 马萧萧, 邓裕宣, 梅迪, 刘磊, 王会岩   

  1. 吉林医药学院检验学院生物技术教研室, 吉林 吉林 132013
  • 收稿日期:2015-12-31 发布日期:2016-07-20
  • 通讯作者: 王会岩,副教授,硕士研究生导师(Tel:0432-64560324,E-mail:zswhy518@163.com) E-mail:zswhy518@163.com
  • 作者简介:刘微(1985-),女,吉林省吉林市人,讲师,理学博士,主要从事生物技术药物方面的研究。
  • 基金资助:

    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)

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

LIU Wei, YAO Yang, MA Xiaoxiao, DENG Yuxuan, MEI Di, LIU Lei, WANG Huiyan   

  1. Department of Biotechnology, Academy of Laboratory, Jilin Medical College, Jilin 132013, China
  • Received:2015-12-31 Published:2016-07-20

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pET22b-SUMO-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40000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1.0 g·L-1、诱导时间为3 h、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。

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

Abstract:

Objective: To design the small ubiquitin modification-fibroblast growth factor receptor 4 (SUMO-FGFR4) 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 was 1.0 g·L-1,the induction time was 3 h,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.

Key words: small ubiquitin-like modifier, fibroblast growth factor receptor 4, recombinant fusion proteins

中图分类号: 

  • Q78