CAJ | 학술논문

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。
목적：연구Pot1단백적은정성。방법：이용만병독표체재체구건표체외원Flag-Pot1적HeLa세포은정극륭，재해극륭중가입CHX억제신단백적합성，검측외원Pot1적반쇠기，학정Pot1적은정성；장Pot1질립여Ub질립공전293T세포후，가입MG132조지단백매체도경，학정Pot1적은정성시부수범소화수식적영향；구건점돌변화절단돌변적Pot1질립，여Ub질립공전293T세포후，가입MG132조지단백매체도경，학정영향Pot1은정성적구역。결과여결론：Pot1통과범소화수식영향자신은정성；점돌변화절단돌변실험증실Pot1존재다개범소화위점，차주요발생재C단400~634위안기산잔기。
Objective: To study the stability of Pot1. Methods: The stability of Pot1 expression, the half life pe?riod 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 stabil?ity 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.