致病蛋白Htt-exon1在大肠杆菌中表达及铜离子结合能力的检测  

Prokaryotic expression of Htt-exon1 in Escherichia coli and the detection of its copper-binding ability

作　　者：项玲 肖桂然 XIANG Ling;XIAO Guiran(School of Food and Biological Engineering, Hefei University of Technology, Hefei 230601, China)

机构地区：[1]合肥工业大学食品与生物工程学院,安徽合肥230601

出　　处：《合肥工业大学学报（自然科学版）》2022年第1期119-124,共6页Journal of Hefei University of Technology：Natural Science

基　　金：国家自然科学基金资助项目(31671284)。

摘　　要：含有过度延长的氨基末端谷氨酰胺重复序列(polyglutamine,polyQ)的Htt-exon1具有神经毒性,导致亨廷顿舞蹈症(Huntington’s disease,HD),研究表明HD病人脑内有过度积累的铜离子,但机制不明。为了探索铜离子与Htt-exon1的关系,文章利用基因工程技术,聚合酶链式反应(polymerase chain reaction,PCR)扩增出含有10个polyQ的Htt-exon1 polyQ10和含有42个polyQ的毒性蛋白Htt-exon1 polyQ42,克隆至pGEX-6p-2原核表达载体上,在大肠杆菌BL21中表达,异丙基-β-D-硫代半乳糖(IPTG)诱导表达出融合蛋白GST-Htt-exon1并分离纯化出Htt-exon1 polyQ10和Htt-exon1 polyQ42,样品硝化后用电感耦合等离子体质谱(inductively coupled plasma masss pectrometry,ICP-MS)检测Htt-exon1结合铜离子的量。结果表明:分离纯化后的GST-Htt-exon1 polyQ10分子量为40 kDa左右,GST-Htt-exon1 polyQ42分子量为50 kDa左右,经PP酶切割掉GST标签后,通过Western Blot进一步验证,纯化后的Htt-exon1 polyQ10分子量为8.5 kDa,Htt-exon1 polyQ42分子量为12.5 kDa,与预期一致;并利用ICP-MS检测发现,Htt-exon1 polyQ10和Htt-exon1 polyQ42均具有结合铜离子的能力。Htt-exon1 containing an excessively long amino-terminal polyglutamine(polyQ)repeat is neurotoxic and leads to Huntington’s disease(HD).The disruption of copper(Cu^(2+))homeostasis is found in HD patients for many years,but the underlying mechanism is still unknown.In order to explore the relationship between copper ion and Htt-exon1,Htt-exon1 polyQ10 and Htt-exon1 polyQ42 were generated by polymerase chain reaction(PCR),and then they were cloned into pGEX-6p-2 for prokaryotic expression.The vector was expressed in Escherichia coli BL21,and the fusion protein GST-Htt-exon1 polyQ was expressed when the E.coli was induced by IPTG.After the sample was nitrated,the amount of fusion protein Htt-exon1 bound to copper ion was detected by inductively coupled plasma mass spectrometry(ICP-MS).The results showed that the molecular weight of GST-Htt-exon1 polyQ10 was about 40 kDa,and that of GST-Htt-exon1 polyQ42 was about 50 kDa.After the GST label was cut off by PP enzyme,Western Blot results showed that the purified Htt-exon1 polyQ10 was about 8.5 kDa,and that of Htt-exon1 polyQ42 was about 12.5 kDa,which were consistent with the expectation;further study indicated that both Htt-exon1 polyQ10 and Htt-exon1 polyQ42 had the ability to bind copper ion.

关 键 词：亨廷顿蛋白 大肠杆菌 原核表达 分离纯化 铜离子结合能力 

分 类 号：Q789[生物学—分子生物学]