期刊论文

中文

医学分子生物学杂志

1672-8009

2008

5

1

35-39

10.3870/j.issn.1672-8009.2008.01.008

湖北省自然科学基金（NO.2005ABA092）~~；

本校为其他机构

目的 构建含VLDL-R配体结合结构域融合蛋白表达载体,并在大肠埃希菌中进行表达与纯化.方法 利用PCR技术扩增LBR1-8的DNA片段,将其克隆至原核表达载体pET28a (+)中,随后将阳性重组质粒转化到大肠埃希菌Rosetta中,利用IPTG诱导蛋白表达并优化其表达条件,表达产物经Ni+-NTA亲和层析柱纯化回收,并采用SDS-PAGE和Western 印迹对目的 蛋白进行分析和鉴定.结果 构建的重组表达质粒经PCR,酶切和DNA测序鉴定与预期的结果一致,含有重组质粒的表达宿主经过IPTG诱导成功表达了LBR1-8,并经优化确定了最佳的诱导表达条件.结论 成功构建pET28a (+)-LBR1-8表达质粒,表达并经纯化得到了LBR1-8.

Objective To construct the expression vector of VLDL-R＇s ligand binding domain fusion protein and study its expression and purification in E. coli. Methods The DNA fragment of LBR1-8 was amplified from VLDL-R cDNA recombinant plasmid by PCR and was inserted into plasmid pET28a （ ＋ ） to construct the prokaryotic expression plasmid pET28a （ ＋ ） -LBR1-8. The positive recombinant plasmid was transformed into E. coli strain Rosetta and then was induced by IPTG, with the expression conditions optimized. The fusion protein was purified by Ni^＋...