截断型线粒体凋亡诱导因子相互作用蛋白质的筛选与鉴定

Identification of Proteins Interacting With Interceptive AIF

线粒体凋亡诱导因子(AIF)是一类存在于线粒体内外膜之间保守的黄素蛋白,它从线粒体到细胞核的转位可诱导细胞凋亡.AIF诱导细胞凋亡的确切机制还不是很清楚.为了筛选可能与截断型AIF存在相互作用的蛋白质,探讨截断型AIF诱导细胞凋亡的机制,用逆转录PCR扩增剪切掉线粒体定位信号的人AIF基因片段并克隆入pcDNA3.1载体,导入肝癌HepG2细胞.成功检测到截断型在转录与翻译水平上的表达.提取转染截断型AIF基因的细胞的蛋白质,用免疫沉淀方法分离与截断型可能存在相互作用的蛋白质,用基质辅助激光解析电离飞行时间质谱对分离得到的蛋白质进行鉴定,其中一蛋白质条带经鉴定为β-actin.再次用反向免疫共沉淀法与Western blot法对筛选出的可能相互作用蛋白质进行验证.结果表明,截断型AIF与β-actin之间可能存在相互作用.

Apoptosis-inducing factor （AIF）, normally confined to mitochondria, can move from mitochondria to nuclei, participate in the execution of cell death. The mechanism by which AIF induces cell apoptosis is unclear. In order to analyze proteins which may interact with AIF, interceptive AIF gene （mitochondrial localization sequence, MLS, was deleted） which was amplified from the total mRNA extracted from hepatoma HepG2 cells by reverse transcriptase polymerase chain reaction（RT-PCR）, was inserted into pcDNA3.1 plasmid.The recombinant plasmids constructed successfully were then transfected into HepG2 cells and expressions of interceptive AIF were detected by RT-PCR and Western blot. Immunoprecipitation （IP） and matrix assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS） were used to isolate and identify proteins which can interact with interceptive AIF respectively. Screened out proteins which may interact with AIF were then confirmed by CO-IP and Western blot. The result showed that AIF may interact with β-actin.