重组蛋白TAT-NLS-Nkx6.2在大肠杆菌的表达纯化及活性测定

中国生物工程杂志

2013, Vol. 33

Issue (9): 24-30

研究报告

重组蛋白TAT-NLS-Nkx6.2在大肠杆菌的表达纯化及活性测定

孙少飞, 汪蓓蕾, 袁婷, 张斌, 张欣, 郭刚, 张瑞

天津医科大学代谢病医院内分泌研究所卫生部激素与发育重点实验室天津 300070

Expression and Fusion Protein TAT-NLS-Nkx6.2 in E.coli and Its Purification and Biological Analysis

SUN Shao-fei, WANG Bei-lei, YUAN Ting, ZHANG Bing, GUO Gang, ZHANG Ru

Institute of Endocrinology/Metabolic Disease Hospital of Tianjin Medical University, Tianjin 300070, China

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摘要： 目的:构建重组基因 TAT-NLS-Nkx6.2 的原核表达载体,获得重组 TN-Nkx6.2 蛋白,并验证其生物学活性。方法:该实验先将人类免疫缺陷病毒HIV-1的反式激活因子TAT、核定位信号 NLS、Nkx6.2基因片段连接合成目的基因TAT-NLS-Nkx6.2 。之后构建重组质粒pET-28a- TN-Nkx6.2 并将其转化入E.coli DH5α中克隆,再转化至E.coli BL21(DE3)中用IPTG诱导表达,经组氨酸Ni²⁺亲和层析纯化融合蛋白,并用SDS-PAGE及Western blot鉴定融合蛋白,荧光免疫组化染色观察TN-Nkx6.2在小鼠脑组织分布。结果:成功表达并获得纯度90%以上的TN-Nkx6.2蛋白,经 Western Blot鉴定,TN-Nkx6.2有良好的免疫原性和免疫反应性荧光免疫组化染色证明TN-Nkx6.2与小鼠大脑细胞结合并内化进入胞浆及胞核。结论:获得的融合蛋白TN-Nkx6.2具有生物学活性,并能穿过血脑屏障与小鼠大脑细胞结合,并内化进入胞核和胞浆,为深入探讨同源盒基因Nkx6.2在甲状腺减低模型的分子机制研究奠定了物质基础。

关键词： Nkx6.2; 重组蛋白; 蛋白纯化; 活性检测

Abstract: Objective:To obtain a fusion protein TN-Nkx6.2, which Nkx6.2 links with TAT and NLS in a recombinant prokaryotic system and to analysis its bioactivity. Methods:In this research, we engineer human immunodeficiency virus HIV-1 trans-activating factor TAT and nuclear location sequence (NLS) into transcription factor Nkx6.2 protein ligate to synthetic gene TAT-NLS-Nkx6.2, recombinant plasmid pET-28a- TN-Nkx6.2 and transform into E.coli BL21 (DE3) for expression by IPTG induction.Then by SDS-PAGE and Western Blot to to identified fusion protein,which purified by histidine Ni²⁺ affinity chromatography.Results:TN-Nkx6.2 was with above 90% purity and good solubility was obtained in vitro,which can combine with brain cells in mice and internalize into the cytoplasm and nucleus proved by fluorescent immunohistochemical.Conclusion:Obtain the fusion protein of TN-Nkx6.2 has biological activity, and can cross the blood-brain barrier into the mice brain cells in nucleus cytoplasm, which lay the material foundation for further explortion of the molecular mechanism that Nkx6.2 hox genes in thyroid reduced model role.

Key words: Homeobox gene Nkx6.2 Protein expression Protein purification Determination of biological activity

收稿日期: 2013-07-03 出版日期: 2013-09-25

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通讯作者: 孙少飞ssfqp@126.com E-mail: ssfqp@126.com

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引用本文:

孙少飞, 汪蓓蕾, 袁婷, 张斌, 张欣, 郭刚, 张瑞. 重组蛋白TAT-NLS-Nkx6.2在大肠杆菌的表达纯化及活性测定[J]. 中国生物工程杂志, 2013, 33(9): 24-30.

SUN Shao-fei, WANG Bei-lei, YUAN Ting, ZHANG Bing, GUO Gang, ZHANG Ru. Expression and Fusion Protein TAT-NLS-Nkx6.2 in E.coli and Its Purification and Biological Analysis. China Biotechnology, 2013, 33(9): 24-30.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I9/24

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