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

作　　者：刘莉[1] 李岳亭 张萌萌[2] 杨予涛[1] 徐志卿[1] 

机构地区：[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

出　　处：《生物技术通报》2015年第2期222-227,共6页Biotechnology Bulletin

基　　金：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)

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c DNA为模板,利用PCR扩增带有Bam H 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p GEX-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Magne GST 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p GEX-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Magne GST 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。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.

关 键 词：锌指结构域 原核表达 蛋白纯化 

分 类 号：Q78[生物学—分子生物学]