sTRAIL蛋白原核表达载体的构建、表达及抗肿瘤活性研究

doi:10.13523/j.cb.20151201

中国生物工程杂志

2015, Vol. 35

Issue (12): 1-7 DOI: 10.13523/j.cb.20151201

研究报告

sTRAIL蛋白原核表达载体的构建、表达及抗肿瘤活性研究

王世奇, 刘婧莹, 刘晨浪, 李纯, 胡晓凤, 夏立秋, 张友明

湖南师范大学生命科学学院微生物分子生物学国家重点实验室培育基地长沙 410081

Construction and Expression of Prokaryotic Expression Vector of Soluble TNF-related Apoptosis Inducing Ligand and Its Anti-tumor Activity

WANG Shi-qi, LIU Jing-ying, LIU Cheng-lang, LI Chun, HU Xiao-feng, XIA Li-qiu, ZHANG You-ming

State Key Laboratory Breeding Base of Molecular Microbial Biology, College of Life Science, Hunan Normal University, Changsha 410081, China

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摘要：

目的:从HL-60细胞中获得了sTrail基因片段,优化蛋白表达条件,并研究其抗肿瘤活性。方法:培养HL-60细胞,提取总RNA,通过RT-PCR扩增sTRAIL蛋白基因片段,并将目的基因克隆至原核表达载体pET28a上,并电击转化E.coli BL21(DE3),IPTG诱导表达,优化蛋白表达条件,Ni-IDA柱纯化重组蛋白,SDS-PAGE蛋白电泳,胶内酶解质谱鉴定。纯化后的重组蛋白作用HUVEC,HeLa,Hep-3B,HCT-116,MDA-MB-231,H460细胞检测蛋白生物学作用。结果:DNA测序结果证实成功构建了重组质粒pET28a-sTrail,SDS-PAGE蛋白电泳,胶内酶解质谱检测显示成功表达sTRAIL蛋白,MTT法和流式细胞术结果显示,sTRAIL蛋白对肿瘤细胞包括HeLa, HCT-116,MDA-MB-231,H460,Hep-3B细胞有良好的生物活性,对正常的HUVEC细胞无毒性。结论:成功构建可以高效表达sTRAIL蛋白的原核表达载体,优化蛋白的表达和纯化后所得sTRAIL蛋白具有良好的抗肿瘤生物活性,为研究和发展利用sTRAIL蛋白作为临床治疗抗肿瘤药物提供了重要基础。

关键词： 基因克隆; 蛋白纯化; 肿瘤坏死因子相关凋亡配体; 原核表达; 抗肿瘤活性

Abstract:

Purpose:To study the induced expression condition and anti-tumor activity of protein sTRAIL from human HL-60 cell. Methods:Genomic RNA was extract from the human HL-60 cell. The sTrail gene segment was amplified with RT-PCR and cloned into the prokaryotic expression vector pET28a. The final expression vector pET28a-sTrail was transformed into E.coli BL21(DE3) for sTRAIL expression. Through IPTG induced, SDS-PAGE and LC-MS were used to determine the correct expression of sTRAIL. After purification by nickel-affinity chromatography, the recombinant sTRAIL was used to HUVEC, HeLa, Hep-3B, HCT-116, MDA-MB-231 and H460 cells. Result:The recombinant plasmid pET28a-sTrail was successfully built by the analysis of DNA sequencing. The result of SDS-PAGE show that sTRAIL gene was successfully expressed in E.coli BL21(DE3) and identified by LC-MS. The purified sTRAIL inhibited the growth of HeLa, HCT-116, MDA-MB-231, H460 and Hep-3B cell while not HUVEC. Flow cytometry showed the inducing apoptosis of Hep-3B cells by sTRAIL. Conclution:The prokaryotic expression vector of sTRAIL was successfully built, and the purified protein sTRAIL exhibited anti-tumor activity. This provided an important foundation for using sTRAIL protein as a antitumor drugs of clinical treatment.

Key words: TNF-related apoptosis inducing ligand Prokaryotic expression Protein purification Antineoplastic activity Gene cloning

收稿日期: 2015-04-15 出版日期: 2015-12-22

ZTFLH:

Q819

基金资助:

国家国际合作项目(2011DFA32610)、国家"973"计划专项(2012CB722301)、国家"863"计划(2011AA10A203)、湖南省协同创新中心项目(20134486)资助项目

通讯作者: 张友明 E-mail: zhangyouming@sdu.edu.cn

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引用本文:

王世奇, 刘婧莹, 刘晨浪, 李纯, 胡晓凤, 夏立秋, 张友明. sTRAIL蛋白原核表达载体的构建、表达及抗肿瘤活性研究[J]. 中国生物工程杂志, 2015, 35(12): 1-7.

WANG Shi-qi, LIU Jing-ying, LIU Cheng-lang, LI Chun, HU Xiao-feng, XIA Li-qiu, ZHANG You-ming. Construction and Expression of Prokaryotic Expression Vector of Soluble TNF-related Apoptosis Inducing Ligand and Its Anti-tumor Activity. China Biotechnology, 2015, 35(12): 1-7.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20151201 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I12/1

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