GST-β-catenin融合蛋白表达载体的构建及其在原核细胞中的表达  

Construction of GST-β-catenin fusion protein expression vectors and the expression in prokaryotic cell

作　　者：梁峰[1] 沈涛[1] 姚强[1] 贾长青[1] 

机构地区：[1]中国医科大学附属盛京医院骨一科,辽宁沈阳110004

出　　处：《解剖学研究》2015年第2期104-107,共4页Anatomy Research

基　　金：国家自然科学基金资助项目(31201053)

摘　　要：目的构建GST-β-catenin融合蛋白表达载体,并在大肠埃希菌(E.col)中诱导表达。方法从COS-7细胞中提取m RNA,反转录为c DNA。用PCR方法扩增出β-catenin基因全长,通过Bam HI和Sal I酶切位点将其定向插入p GEX-4T-2载体中,构建原核表达质粒p GEX-4T-2-β-catenin,并转化E.coli DH5α,通过限制性内切酶酶切电泳和DNA测序鉴定正确后,转入E.coli BL21中,经异丙基硫代β-D半乳糖苷大量诱导表达,SDS-PAGE电泳和Western blot鉴定。结果酶切电泳及测序结果证明,成功构建了原核表达质粒GST-β-catenin,并用Western blot方法证实了GST-β-catenin融合蛋白的表达。结论成功构建了GST-β-catenin原核表达载体,并证实了其在原核细胞大肠埃希菌中的表达,为进一步纯化β-catenin及研究其结构与功能提供了前提基础。Objective To construct GST-β-catenin fusion protein expression vector and induce its expression in Escherichia coli（E.coli）. Methods Total m RNA was extracted from COS-7 cells, and c DNA was formed by reverse transcription.Theβ-catenin coding sequence was amplified by polymerase chain reaction（PCR） and subcloned into p GEX-4T-2 vector. The positive recombinant was identified by restriction enzyme digestion and DNA sequencing. Then they were transformed into E.coli BL21, induced by IPTG and identified by SDS-PAGE and Western blot. Results The prokaryotic expression plasmid p GEX-4T-2-β-catenin was successfully constructed and confirmed by enzyme digestion and sequencing. The GST- β-catenin fusion proteins were expressed and confirmed by Western blot. Conclusion The prokaryotic expression plasmid ofβ-catenin was successfully constructed and the expression of fusion proteins in E.coli was confirmed. This study provides the basis for the further research on purifying Vinexin protein and the biological function ofβ-catenin.

关 键 词：Β-CATENIN 原核表达 融合蛋白 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]