天花粉蛋白突变体的构建及其与重组天花粉在原核系统中的表达和纯化  

Expression and purification of mutant trichosanthin in escherichia coli

作　　者：曹卫红[1] 黄利鸣[1] 王艳林[1] 刘朝奇[1] 彭平平[1] 宋华梅[1] 尤程程[1] 黄益玲[1] 谭寒星[1] 吴燕珍[1] 

机构地区：[1]三峡大学医学院分子生物学研究所,宜昌443002

出　　处：《中华中医药杂志》2011年第1期80-83,共4页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金项目(No.30873282);2009年湖北省自然科学基金计划项目(No.2009CDA060)~~

摘　　要：目的:对天花粉蛋白(TCS)的第120-123位氨基酸进行缺失突变,并在原核系统中对其进行表达和纯化。方法:利用重叠延伸PCR的方法得到TCS突变体cDNA,并克隆入原核表达载体pET-28(a+)。酶切和测序鉴定后,将突变体质粒pET-28(a+)-mTCS转化BL21(DE3)表达菌,经IPTG诱导表达后,对表达产物用Ni2+-NTA亲和层析柱进行纯化和Western-blot鉴定。结果:利用重叠延伸PCR成功获得突变天花粉蛋白的编码序列,并构建pET-28(a+)-mTCS原核表达质粒。在BL21(DE3)菌中,突变体蛋白(mTCS)可被IPTG诱导性表达,并被Ni2+-NTA树脂有效纯化。结论:本实验成功获得一种突变体TCS,并建立该突变TCS的原核表达和纯化的实验方法。Objective： To construct a mutated trichosanthin（mTCS） in which four anino acids（Aa120-123） are deleted and to express and purify this mTCS in E.coli.Methods： cDNA encoding mTCS was obtained by PCR-driven overlap extension,and then cloned into pET-28a（＋） vector.After restriction and sequence analysis,the recombinant plasmid pET-28a（＋）-mTCS was transformed into BL21（DE3）.The mTCS was expressed by IPTG induction and purified by Ni-NTA resin.Results： mTCS cDNA was obtained and cloned into the pET-28a（＋） vector successfully.In BL21（DE3）,the mTCS was expressed by IPTG induction,and purified by Ni-NTA resin efficiently.Conclusion： A mutated TCS was obtained in this study and a technique method for prokaryotic expression and purifaication of this mutated TCS was established successfully.

关 键 词：天花粉蛋白 基因突变 原核表达 

分 类 号：R285[医药卫生—中药学]