一种新型的截短型转化生长因子βⅡ型受体在大肠杆菌中的表达及活性鉴定

中国生物工程杂志

2009, Vol. 29

Issue (08): 33-37

研究报告

一种新型的截短型转化生长因子βⅡ型受体在大肠杆菌中的表达及活性鉴定

初彦辉¹,刘海峰²,王小花³,张羽飞³,武艳³,袁晓环³

1. 牡丹江医学院黑龙江省抗纤维化生物治疗重点实验室牡丹江 157011
2. 暨南大学生命科学与技术学院广州 5106323
3.牡丹江医学院病原生物学实验室牡丹江 157011

Expression and Characterization of a Novel Truncated TGF-&beta|Receptor II Synthesized in Escherichia coli

CHU Pan-Hui^1,2**, LIU Hai-Feng¹, WANG Xiao-Hua³, ZHANG Hu-Fei¹, WU Yan¹, YUAN Xiao-Huan¹

1.Heilongjiang Key Laboratory of Antifibrosis Biotherapy, Mu Dan Jiang Medical University, Mudanjiang157011, China
2.College of Life Science and Technology, Jinan University, Guangzhou510632,China
3.The Laboratory of Pathogeny Biology, Mu Dan Jiang Medical University, Mudanjiang157011, China

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摘要：

克隆人源的截短型转化生长因子βⅡ型受体（tTGF-βRⅡ），成功构建高效稳定的大肠杆菌表达菌株。采用PCR技术扩增得到目的基因tTGF-βRⅡ，插入原核表达载体pGEX4T3的相应位点，并在大肠杆菌BL21（DE3）中获得高可溶性表达的重组蛋白，重组蛋白GST-tTGF-βRⅡ经Glutathione-Sepharose 4B亲和层析纯化后，再经凝血酶酶切，然后经Glutathione-Sepharose 4B纯化获得目的蛋白tTGF-βRⅡ，SDS-PAGE分析表明：可以通过Glutathione-Sepharose 4B亲和层析纯化得到GST-tTGF-βRⅡ，其分子量约37.0 KDa，和目的蛋白tTGF-βRⅡ，分子量为11.0 KDa。Western blot结果显示GST-tTGF-βRⅡ和tTGF-βRⅡ为特异性蛋白。运用基因工程的方法获得tTGF-βRⅡ目的蛋白，对瘢痕成纤维细胞增殖具有很好抑制作用。

关键词： 转化生长因子βⅡ型受体; 瘢痕成纤维细胞; 表达; 纯化

Abstract:

To clone human truncated TGF-β receptor II（tTGF-βRⅡ）and express it in Escherichia coli. The gene tTGF-βRⅡ,obtained by PCR was cloned into the vector pGEX4T3 to construct a fusion expression plasmid.The recombinant protein GST-tTGF-βRⅡ were expressed in soluble form, purified by Glutathione-Sepharose 4B affinity chromatography and cleaved by thrombin to release tTGF-βRⅡ. Purification of tTGF-βRⅡ was achieved by affinity chromatography. SDS-PAGE indicated that fusion protein GST- tTGF-βRⅡ was secreted as a protein of around 37.0 KDa and aimed protein tTGF-βRⅡwas 11.0 KDa. The GST-tTGF-βRⅡand tTGF-βRⅡ protein were confirmed by Western blot analysis. The purification of aimed protein was biologically active in a validated inhibited human fibroblast proliferation bioassay.

Key words: tTGF-βRⅡ fibroblast expression purification

收稿日期: 2009-03-26 出版日期: 2009-07-28

ZTFLH:

R392.11

基金资助:

国家“863”计划（2006AA020906）、黑龙江省自然科学基金（D200799）资助项目

通讯作者: 初彦辉

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引用本文:

初彦辉,刘海峰,王小花,张羽飞,武艳,袁晓环. 一种新型的截短型转化生长因子βⅡ型受体在大肠杆菌中的表达及活性鉴定[J]. 中国生物工程杂志, 2009, 29(08): 33-37.

CHU Pan-Hui, LIU Hai-Feng, WANG Xiao-Hua, ZHANG Hu-Fei, WU Yan, YUAN Xiao-Huan. Expression and Characterization of a Novel Truncated TGF-&beta|Receptor II Synthesized in Escherichia coli. China Biotechnology, 2009, 29(08): 33-37.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I08/33

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