人Pokemon蛋白锌指结构域的表达与纯化

doi:10.13560/j.cnki.biotech.bull.1985.2015.02.033

生物技术通报 ›› 2015, Vol. 31 ›› Issue (2): 222-227.doi: 10.13560/j.cnki.biotech.bull.1985.2015.02.033

人Pokemon蛋白锌指结构域的表达与纯化

刘莉¹, 李岳亭¹, 张萌萌², 杨予涛¹, 徐志卿¹

1.首都医科大学基础医学院，北京 100069;
2.嘉兴出入境检验检疫局，嘉兴 314001

收稿日期:2014-08-08 出版日期:2015-02-05 发布日期:2015-02-06
作者简介:刘莉，女，硕士研究生，研究方向：神经生物学；E-mail：emil_lily@163.com
基金资助:
国家“973”计划（2010CB912003），国家自然科学基金项目（31271154，31171032），高等学校博士学科点专项科研基金项目（20111107120011），北京市教委科技发展计划（KM201310025001）

Prokaryotic Expression and Purification of Zinc Finger Domain of Human Pokemon Protein

Liu Li¹, Li Yueting¹, Zhang Mengmeng², Yang Yutao¹, Xu Zhiqing¹

1. School of Basic Medical Sciences, Capital Medical University, Beijing 100069； 2. Jiaxing Entry-Exit Inspection and Quarantine, Jiaxing 314001

Received:2014-08-08 Published:2015-02-05 Online:2015-02-06

摘要/Abstract

摘要： 旨在原核表达Pokemon基因的锌指结构域，纯化获得GST-Zinc finger的融合蛋白。以人胶质瘤T98G细胞的cDNA为模板，利用PCR扩增带有BamH I和Sal I 酶切位点的人Pokemon基因的锌指结构域，然后将其克隆到pGEX-4T-1原核表达载体中。将正确的重组载体转入大肠杆菌BL21（DE3），用IPTG诱导表达，再利用MagneGST particles亲和纯化Zinc finger融合蛋白，最后通过Western blot鉴定此融合蛋白。结果显示，成功构建pGEX-4T-1-Zinc finger原核表达载体；30℃条件下，0.2 mmol/L的IPTG能诱导出大量的可溶性GST-Zinc finger蛋白；经MagneGST particles纯化的GST-Zinc finger蛋白可被识别Pokemon锌指结构域的抗体特异识别。纯化的GST-Zinc finger蛋白可用于后续的生物学研究。

关键词: Pokemon, 锌指结构域, 原核表达, 蛋白纯化

Abstract: It was to express human Zinc finger domain of Pokemon gene in prokaryotic cells and purify the GST-Zinc finger fusion protein. The segment of zinc finger domain of Pokemon gene was amplified by PCR and cloned into prokaryotic expression vector pGEX-4T-1. The recombinant plasmid pGEX-4T-1-Zinc finger was transformed into E.coli BL21（DE3）and exogenous protein was induced by IPTG. After purification using MagneGST particles, the GST-Zinc finger fusion protein was further identified by Western blot. Results showed that the recombinant plasmid pGEX-4T-1-Zinc finger was constructed successfully. When BL21（DE3） cells transformed with pGEX-4T-1-Zinc finger were cultured at 30℃ and induced with 0.2 mmol/L IPTG, GST-Zinc finger protein was obtained in a large quantity in supernatant. The purified GST-Zinc finger was further identified specifically by Pokemon antibody. Therefore, it proved that GST-Zinc finger fusion protein was successfully expressed and purified, and could be used for further study of the function of Pokemon.

Key words: Pokemon, Zinc finger, prokaryotic expression, protein purification

刘莉, 李岳亭, 张萌萌, 杨予涛, 徐志卿. 人Pokemon蛋白锌指结构域的表达与纯化[J]. 生物技术通报, 2015, 31(2): 222-227.

Liu Li, Li Yueting, Zhang Mengmeng, Yang Yutao, Xu Zhiqing. Prokaryotic Expression and Purification of Zinc Finger Domain of Human Pokemon Protein[J]. Biotechnology Bulletin, 2015, 31(2): 222-227.

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人Pokemon蛋白锌指结构域的表达与纯化

Prokaryotic Expression and Purification of Zinc Finger Domain of Human Pokemon Protein

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