线粒体锚定对凋亡诱导因子抗氧化应激功能的影响  

Apoptosis-Inducing Factor as an Antioxidant is Dependent on An choring on Mitochondria

作　　者：凌丽君[1,2] 刘萱[2] 曹诚[2] 

机构地区：[1]中南大学生命科学与技术学院,湖南长沙410000 [2]军事医学科学院生物工程研究所,北京100850

出　　处：《生物技术通讯》2013年第4期453-456,共4页Letters in Biotechnology

基　　金：国家自然科学基金重点项目(30730027/C050102)

摘　　要：目的:构建带线粒体锚定信号肽的凋亡诱导因子(AIF)融合表达载体,研究在A549细胞中AIF线粒体锚定与其抗氧化应激功能的关系。方法:利用PCR将AIF原有线粒体定位信号(1-120 aa)替换成具有锚定功能的细胞色素c氧化酶Ⅳ亚型(COXⅣ)线粒体定位信号,并将COX-AIF克隆至pEGFP-N1和pDsRed1-N1载体,构建COX-AIF-GFP和COX-AIF-RFP融合表达载体;利用免疫印迹和激光共聚焦技术检测COX-AIF-GFP和COX-AIF-RFP的表达及其与线粒体的共定位;利用DCF染色和流式细胞技术检测A549细胞内过氧化物的水平。结果:表达了COX-AIF-GFP和COX-AIF-RFP融合蛋白,COX-AIF-GFP/RFP及AIF-RFP/RFP均定位于线粒体;与野生型AIF-RFP相比,COX-AIF-RFP可显著提高A549细胞的抗氧化应激能力。结论:AIF抗氧化应激能力依赖其在线粒体内膜的锚定。Objective： The mitochondria-anchored apoptosis-inducing factor（AIF） with a florescence tag was con-structed to discover the effect of mitoehondrial-loealization on the anti-oxidative stress activity of AIF. Methods： The original mitoehondrial localization signal（MLS） of AIF（1-120 aa） was replaced by the MLS of cytochrome c oxidase subunitⅣ （COX Ⅳ ）, which could not be dissociated from mitochondria. Then COX-AIF fragment was cloned into pEGFP-N1 and pDsRedl-N1 vector, respectively. The expression of COX-AIF-GFP/RFP and their co-localization with mitochondria.were detected by immunoblotting and confocal. The ROS levels of cells transfect- ed with AIF-RFP or COX-AIF-RFP were analyzed by DCF staining and flow cytometry. Results： COX-AIF-GFP/ RFP fusion proteins were successfully expressed and co-localized with mitochondria in 293T cells. Comparing with wild type AIF-RFP, COX-AIF-RFP manifested significantly enhanced anti-oxidative stress activity in A549 cells. Conclusion： The anti-oxidative stress activity of AIF is dependent on its mitoehondria-anehoring ability.

关 键 词：凋亡诱导因子 线粒体 氧化应激 

分 类 号：Q78[生物学—分子生物学]