截短的食管癌相关基因ECRG4在大肠杆菌中的可溶性表达及纯化  

The Soluble Expression and the Purification of Cut-Short Esophageal Cancer Related Gene ECRG4 in Escherichia Coli

作　　者：杨扬[1] 樊青霞[1] 王留兴[1] 郭黎平[2] 陆士新[1] 

机构地区：[1]郑州大学第一附属医院肿瘤科郑州大学肿瘤中心,河南郑州450052 [2]中国医学科学院肿瘤研究所病因与癌变研究室,北京100021

出　　处：《肿瘤基础与临床》2006年第3期169-171,共3页journal of basic and clinical oncology

基　　金：国家973重点基础研究项目(编号:2004BC518701)

摘　　要：目的本实验旨在构建ECRG4的原核表达载体,在大肠杆菌中表达并纯化ECRG4所编码的蛋白,为进一步研究奠定基础。方法将ECRG4基因序列前端编码疏水性跨膜区28个氨基酸的cDNA序列截去,再将截短的cDNA序列克隆到原核表达载体Pet30(+)上,重组表达载体转化感受态表达菌株BL21(DE3),低浓度IPTG低温诱导融合蛋白的表达。采用Western blot鉴定目的蛋白的表达,使用Ni-NTA亲和层析柱进行纯化。结果构建了截短的ECRG4-pET30a(+)基因的原核表达载体,IPTG低温诱导得到大量可溶性蛋白,并且通过亲和层析纯化了重组融合蛋白。结论成功获得了高效表达的、具有一定的生物学活性的ECRG4原核表达产物,并为进一步研究ECRG4的结构和功能打下基础。Objective This assay was designed to construct the prokaryotic expression vector, investigate the expression of ECRG4 in E. coil and purify its product. Methods The cut-short eDNA of ECRG4 was inserted into the vector pET30a（ ＋ ）. The recombinant vector was transfected into E coli BL21 （DE3） and induced the expression of this his-fusion protein by low concentration of IPTG and low temperature overnight. After sonification, the supernatants were analyzed by SDS-PAGE and the results were conformed by Western blot analysis and purified by affinity chromatography. Results After induction, a new anticipating fusion protein of 20kD appeared as an expected size, and mainly existed in a soluble form. Conclusions The high expression of the soluble protein in a stable level of 10 - 15 mg/L made it possible to go on further study.

关 键 词：ECRG4融合蛋白 可溶性表达 纯化 增殖抑制 食管癌相关基因 

分 类 号：R735.1[医药卫生—肿瘤]