热休克蛋白60基因定点诱变导致其在细胞内定位的改变  

Site-directed Mutation of Serine70 on HSP60 Subcellular Relocation

作　　者：王银[1,2] 李云鸿[1] 滕鹏[2] 成文静[2] 张玉梅[3] 苗珍花[4] 蒲静[3] 马娇[3] 张楠[3] 游丽琴[3] 

机构地区：[1]宁夏医科大学科学技术中心,银川750004 [2]宁夏医科大学基础医学院神经生物系,银川750004 [3]宁夏医科大学基础医学院医用化学系,银川750004 [4]宁夏医科大学基础医学院人体解剖与组织胚胎学系,银川750004

出　　处：《宁夏医科大学学报》2011年第7期609-611,618,共4页Journal of Ningxia Medical University

基　　金：宁夏医科大学特殊人才项目(XT200910);国家自然科学基金(30960108;31060140;81060034);教育部留学回国人员基金

摘　　要：目的研究将热休克蛋白60(HSP60)蛋白序列中第70位的丝氨酸(serine)突变为丙氨酸(alanine)后,HSP60在细胞内定位的改变。方法将PrC/CMV-HSP60P1重组质粒中HSP60P1编码第70位丝氨酸的碱基定点诱变为编码丙氨酸的碱基序列,转染至小胶质细胞BV-2细胞株中表达。细胞经10h缺氧处理后,裂解细胞,应用差速离心方法分离细胞膜、细胞质和线粒体,应用Western-blot方法检测各细胞器中HSP60的含量。结果在转染未诱变PrC/CMV-HSP60P1质粒的细胞中,正常情况下,HSP60大部分分布在线粒体,少部分在胞浆,细胞膜中未见表达;缺氧刺激后,HSP60在细胞膜、胞浆和线粒体中均有表达;然而70位丝氨酸突变为丙氨酸后,只观察到HSP60在胞浆和线粒体中的表达,而在细胞膜上未见表达。结论作为磷酸化位点的Serine70对HSP60在细胞内的定位具有关键作用。Objective To explore the site-directed mutation of serine70 to alanine on HSP60 subcellular location.Methods Site-directed mutation of serine70 to alanine were performed in the recombinated plasmid PrC/CMV/HSP60P1 and transfected into BV-2 cells.After ten hours hypoxia,cells were broken and mitochondria,cytoplasm and plasma membrane were collected by centrifugation.HSP60 was checked in these parts by western-blot.Results In transfected nonmutated HSP60 cells and in normal condition,HSP60 was not appeared on plasma membrane,however,after hypoxia,HSP60 translocated to plasma membrane.After mutation,HSP60＇s relocation to plasma membrane was abolished.Conclusion Serine70 is a key phosphorylation site for HSP60 subcellular relocation.

关 键 词：HSP60 定点诱变 细胞内的定位 磷酸化位点 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]