大鼠Scratch2蛋白锌指结构域的表达与纯化  

Prokaryotic expression and purification of zinc finger domain of rat Scratch2 protein

作　　者：李岳亭 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

出　　处：《生物技术》2017年第5期440-445,451,共7页Biotechnology

基　　金：国家自然科学基金项目("调节GALR2基因关键转录因子的鉴定及其在抑郁中的作用";No.31271154;"甘丙肽及其受体在深部脑磁刺激抗抑郁过程中的作用机制研究";No.81671345);北京市自然科学基金项目("miR-15b在抑郁症发生中的作用及机制研究";No.7162016)

摘　　要：[目的]原核表达大鼠Scratch2基因的C_2H_2型锌指结构域,纯化获得GST-C_2H_2融合蛋白。[方法]以新鲜大鼠前额叶脑组织cDNA为模板,利用PCR扩增带有EcoRⅠ和XhoⅠ酶切位点的Scratch2基因的锌指结构域后,构建原核表达载体pGEX-4T-1-C_2H_2,并将其转化至大肠杆菌BL21(DE3),经IPTG诱导融合蛋白表达,再利用MagneGST particles亲和纯化GST-C_2H_2融合蛋白,最后通过Western Blot鉴定此融合蛋白。[结果]成功构建了pGEX-4T-1-C_2H_2原核表达载体;在20℃、0.2 mmol/L的IPTG诱导下,GST-C_2H_2融合蛋白即可有效地表达;经MagneGST particle纯化的GST-C_2H_2蛋白可被识别Scratch2锌指结构域的抗体所识别。[结论]纯化的GST-C_2H_2蛋白可用于后续研究。[Objective] To express rat zinc finger domain of Scratch2 gene in prokaryotic cells and purify the GST-C_2H_2 fusion protein. [Methods] The zinc finger domain of Scratch2 gene was amplified by PCR and inserted into prokaryotic expression vector pGEX-4 T-1. The recombinant pGEX-4 T-1-C_2H_2 plasmid was transformed into E. coli BL21（ DE3） and exogenous protein was induced by IPTG. After purification using MagneGST particles,the GST-C_2H_2 fusion protein was further identified by Western Blot analysis. [Results] The recombinant plasmid pGEX-4 T-1-C_2H_2 was constructed successfully.When BL21（ DE3） cells transformed with pGEX-4 T-1-C_2H_2 were cultured at 20℃ and induced with 0. 2 mmol/L IPTG,GST-C_2H_2 fusion protein was expressed efficiently. In addition,the purified GST-C_2H_2 was further identified specifically by Scratch2 antibody. [Conclusion] The GST-C_2H_2 fusion protein was successfully expressed and purified,and it could be used to further study the function of Scratch2.

关 键 词：Scratch2 锌指结构域 原核表达 蛋白纯化 

分 类 号：Q786[生物学—分子生物学]