Pot1通过泛素化修饰影响自身的稳定性  

The Stability of Pot1 Affected by the Ubiquitinytion

作　　者：杨丹丹[1,2] 金蕊[2] 张金丁 王亚楠[1,3] 黄君健[2] 魏道智[1] 

机构地区：[1]福建农林大学生命科学学院,福建福州350002 [2]军事医学科学院生物工程研究所,北京100850 [3]沈阳农业大学生物科学技术学院,辽宁沈阳110866

出　　处：《生物技术通讯》2015年第2期170-173,共4页Letters in Biotechnology

基　　金：国家自然科学基金(81171916)

摘　　要：目的：研究Pot1蛋白的稳定性。方法：利用慢病毒表达载体构建表达外源Flag-Pot1的HeLa细胞稳定克隆，在该克隆中加入CHX抑制新蛋白的合成，检测外源Pot1的半衰期，确定Pot1的稳定性；将Pot1质粒与Ub质粒共转293T细胞后，加入MG132阻止蛋白酶体途径，确定Pot1的稳定性是否受泛素化修饰的影响；构建点突变和截短突变的Pot1质粒，与Ub质粒共转293T细胞后，加入MG132阻止蛋白酶体途径，确定影响Pot1稳定性的区域。结果与结论：Pot1通过泛素化修饰影响自身稳定性；点突变和截断突变实验证实Pot1存在多个泛素化位点，且主要发生在C端400-634位氨基酸残基。Objective： To study the stability of Pot1. Methods： The stability of Pot1 expression, the half life period of Pot1 expression, in HeLa cells infected retroviruses express Flag-Pot1, was detected after CHX was added to inhibite new proteins expression. In order to validate the relationship between the ubiquitinylation and the stability of Pot1, MG132 was used to prohibit proteasomes after the cotransfection of Pot1 plasmid and Ub plasmid in 293T cells, and then the expression of Pot1 was detected by Western blot. Point mutation and deletion plasmids of Pot1 were constructed, and then were cotransfected in 293T cells with Ub plasmid before MG132 was used to prohibit proteasomes to study definitely the region which affected the stability of Pot1. Results Conclusion：The stability of Pot1 is affected by ubiquitinytion, and the key region is in 400-634 of Pot1 protein sequence.

关 键 词：Pot1 稳定性 泛素化 

分 类 号：Q78[生物学—分子生物学]