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| 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 | |
| 引用本文： | 杨扬,樊青霞,王留兴,郭黎平,陆士新.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[J].肿瘤基础与临床,2006,19(3):169-171. |
| 作者姓名： | 杨扬 樊青霞 王留兴 郭黎平 陆士新 |
| 作者单位： | 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 3. 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;中国医学科学院肿瘤研究所病因与癌变研究室,北京,100021 |
| 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。 |
| 关 键 词： | ECRG4融合蛋白 可溶性表达 纯化 增殖抑制 |
| 文章编号： | 1673-5412（2006）03-0169-03 |
| 收稿时间： | 2006-03-22 |
| 修稿时间： | 2006年3月22日 |
The Soluble Expression and the Purification of Cut-Short Esophageal Cancer Related Gene ECRG4 in Escherichia Coli |
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| Yang Yang,Fan Qingxia,Wang Liuxing,Guo Liping,Lu Shixin.The Soluble Expression and the Purification of Cut-Short Esophageal Cancer Related Gene ECRG4 in Escherichia Coli[J].journal of basic and clinical oncology,2006,19(3):169-171. | |
| Authors: | Yang Yang Fan Qingxia Wang Liuxing Guo Liping Lu Shixin |
| Institution: | 1. Department of Oncology, the First Affiliated Hospital of Zhengzhou University, Tumor Center of Zhengzhou University, Zhengzhou 450052, China; 2. Department of Etiology and Carcinogenesis, Cancer Institute, Chinese Academy of Medical Sciences, Beijing 100021, China |
| Abstract: | Objective This assay was designed to construct the prokaryotic expression vector,investigate the expression of ECRG4 in E.coli and purify its product.Methods The cut-short cDNA 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. |
| Keywords: | ECRG4 fusion protein soluble expression purification reproduction restraint |
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