小同功型人硒蛋白P的诱导凋亡活性  

The apoptosis-inducing activity of human selenoprotein P shorter isoform

作　　者：房青[1] 伊瑶[2] 郑艳华[3] 陈佺[3] 宁力[1] 查园园[1] 毕胜利[2] 杨建国[4] 林晨[1] 

机构地区：[1]中国医学科学院中国协和医科大学肿瘤研究所分子肿瘤学国家重点实验室,北京100021 [2]中国疾病预防控制中心病毒病预防控制所肝炎室 [3]中国科学院动物研究所生物膜与膜生物工程国家重点实验室 [4]北京市营养源研究所

出　　处：《中华肿瘤杂志》2004年第11期652-656,共5页Chinese Journal of Oncology

基　　金：国家自然科学基金资助项目 (3 0 3 70 3 0 2 ) ;北京市自然科学基金资助项目 (70 0 2 0 0 7)

摘　　要：目的　研究小同功型人硒蛋白P(HSelP)的作用。方法　克隆HSelP小同功型基因 ,将位于N端 4 0位的硒半胱氨酸 (SeCys)突变为半胱氨酸 ,测序确定后 ,在感受态大肠杆菌BL2 1中大量表达 ,表达产物HSelP2 80m用阴离子交换层析纯化 ,Westernblot方法鉴定。提取小鼠肝细胞线粒体 ,通过荧光光谱仪检测线粒体悬液 90°光散射强度和罗丹明 12 3荧光光度变化 ,确定不同浓度HSelP2 80m引起的线粒体膜透过性转运孔 (PTP)开放程度和线粒体跨膜电位的变化。结果　成功地将HSelP小同功型中SeCys点突变为半胱氨酸 ,在原核表达系统中获得大量表达。表达产物纯化后纯度达 90 %以上。HSelP2 80m可促进线粒体PTP开放 ,跨膜电位下降 ,且有剂量依赖关系 ,可被PTP的特异抑制剂所抑制。此作用在去除N端 4 0个氨基酸后丧失。结论　HSelP2 80m有促进线粒体PTP开放的作用 ,为进一步研究HSelP的结构和功能提供了线索。Objective Human selenoprotein P (HSelP) is unique protein that contains 10 selenocysteines encoded by 10 inframe UGA, which typically function as stop codon. The function of HSelP remains unclear, in part due to the inability to express it by gene recombinant technique. This study is to investigate expression and purification of recombinant HSelP in prokaryotic expression system, and its activity to induce apoptosis in vitro. Methods The shorter HSelP isoform was cloned. After the selenocysteine (SeCys) at 40 th position from N terminus of the HSelP shorter isoform was mutated into cysteine by PCR, it was expressed in E. coli. The expressed product was purifed with DEAE column and identified by Western blot. Subsequently, its function on induction of mitochondrial apoptotic activity was studied. Results The mutant HSelP shorter isoform expressed in prokaryotic system was purified by DEAE column to 90% homogeneity. The purified product, HSelP280m, induced the opening of mitochondrial permeability transition pore (PTP) and decreased the transmembrane potential in a dose-dependent manner. These events could be abolished by PTP specific inhibitors. ConclusionHSelP280m can induce the opening of mitochondrial PTP, which provides a basis for investigating the structure and function of recombinant HSelP.

关 键 词：人硒蛋白P 诱导 凋亡 活性 氨基酸 

分 类 号：R73-3[医药卫生—肿瘤]