重组蛋白TAT-NLS-Nkx6.2在大肠杆菌的表达纯化及活性测定  

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

作　　者：孙少飞[1] 汪蓓蕾[1] 袁婷[1] 张斌[1] 张欣[1] 郭刚[1] 张瑞[1] 

机构地区：[1]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

出　　处：《中国生物工程杂志》2013年第9期24-30,共7页China Biotechnology

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-TNNkx6.2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TNNkx6.2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。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.

关 键 词：Nkx6.2 重组蛋白 蛋白纯化 活性检测 

分 类 号：Q753[生物学—分子生物学]