J4 ›› 2011, Vol. 37 ›› Issue (4): 656-660.

• 基础研究 • 上一篇    下一篇

重组人胶原蛋白肽在大肠杆菌中的高效表达

陈 光|吴 铭|王 刚 |孙 旸   

  1. 吉林农业大学生命科学院生物物理实验室|吉林 长春 130118
  • 收稿日期:2011-01-06 出版日期:2011-07-28 发布日期:2011-07-28
  • 通讯作者: 陈 光(Tel:0431-84531255,E-mail: chg61@163.com) E-mail:chg61@163.com
  • 作者简介:陈 光(1961-)|女|吉林省长春市人|教授|理学博士|主要从事酶工程和基因工程方面的研究。
  • 基金资助:

    吉林省自然科学基金项目资助课题(20070207);高等学校博士学科点专项科研基金资助课题(2009222311001)

Expression of recombinant human collagen peptide in E.coli

CHEN Guang|WU Ming|WANG Gang|SUN Yang   

  1. Laboratory of Biophysics,College of Life Sciences|Jilin Agricultural University|Changchun 130000|China
  • Received:2011-01-06 Online:2011-07-28 Published:2011-07-28

摘要:

目的:构建含有胶原蛋白基因的原核表达载体pET32a-CP6,并转入大肠杆菌BL21(DE3)中进行高效表达,为获得大量可溶的胶原蛋白肽提供可靠依据。方法:将重组表达载体pET32a-CP6转化到大肠杆菌BL21中,以IPTG为诱导剂,对温度、接种量、诱导时机、IPTG诱导浓度等各种发酵参数进行优化,筛选高效表达的发酵条件,并通过Ni-NTA亲和层析进行纯化分析。结果:菌株在37℃、接种量为2%、摇瓶培养约2.5 h后,加入终浓度为0.5 mmol·L-1的IPTG,37℃诱导5 h,收获的蛋白产量最高为31.52 mg·L-1,Western blotting分析该表达产物与人COL6A2单克隆抗体有特异结合能力。结论:优化工程菌高效表达重组人胶原蛋白,纯化重组蛋白。

关键词: 重组胶原蛋白肽;重组蛋白纯化;大肠杆菌

Abstract:

Abstract:Objective To construct the  recombinant expression vector pET32a-CP6 and  transform  into the Escherichia colicE.coli) BL21 (DE3) and express the recombinant protein at a high level,in order to provide reliable basis for obtaining a large number of soluble collagen. Methods The recombinant plasmid pET32a-CP6 was transformed into E.coli BL21 and induced by IPTG.The expression conditions of the recombinant protein were optimized in this test,which included temperature,time for induction,concentration of ITPG,inoculation volume,adding time of IPTG.The recombinant E.coli was fermented under the optimal condition,and the lysate of bacteria was purified by nickel ion affinity.The purified target protein was determined for purity by SDS-PAGE.Results The optimal temperature and time for induction of recombinant E.coli were 37℃ and 5 h respectively,while the optimal concentration of IPTG as an inducer was 0.5 mmol·L-1.The expression level of target protein reached 31.52 mg·L-1 under the optimal condition.The Western blotting results showed that the recombinant protein had specific reaction with anti-human COL6A2 antibody.Conclusion The optimized recombinant E.coli can efficiently express the recombinant human collagen,the recombinant protein is purified successfully.

 

Key words: recombinant human collagen peptide;purification of recombinant protein;Escherichia coli

中图分类号: 

  • Q78