一个新的端粒重复序列结合蛋白1相互作用蛋白的鉴定及其相互作用的研究

Identification and interaction study of a new protein that interact with telomeric repeat binding factor 1

目的 为了进一步探讨TRF1的生物学功能,应用蛋白质肽指纹谱技术鉴定与TRF1相互作用的新的蛋白质,并对作用蛋白进行相互结合研究.方法 运用蛋白质肽指纹谱技术,对持续稳定表达Flag-TRF1的人HeLa细胞裂解液免疫沉淀产物进行分析,并对得到的外源性片段进行质谱测定及同源性分析.结果 成功构建稳定表达Flag-TRF1的人HeLa细胞,并进行蛋白质表达验证;蛋白质质谱分析得到一个新的TRF1相互作用蛋白β-TrCP;免疫共沉淀实验及体外沉降实验证实β-TrCP能够与TRF1在体内及体外相互结合;免疫荧光实验证实β-TrCP与TRF1在细胞内存在共定位.结论 应用蛋白质肽指纹谱技术,筛选到β-TrCP是一个新的TRF1相互结合蛋白,TRF1与β-TrCP能够在体内、外相互结合,TRF1与β-TrCP在HeLa细胞内存在共定位,为进一步研究TRF1的生物学功能提供了新的线索.

Objective To screen and identify the new interactive protein of TRF1 （telomeric repeat blinding factor 1） using MALDI-TOF mass spectrometry and provide clues for the studies on TRF1 biological function.Methods The immunoprecipitation assay was performed to isolate Flag-TRF1 protein and the protein complex was subjected to MALDI-TOF mass spectrometry for protein identification.The fragments of identified amino acid were analyzed with Blast in NCBI.Results Flag-TRF1 HeLa cell line were stable expressed and constructed.β-TrCP protein was identified from the Flag-TRF1 precipitate.Immunoprecipition experiment and sedimentation test show that β-TrCP could interact with TRF1 in vivo and in vitro; Immunofluorescence study showed that β-TrCP colocalizes with TRF1 in HeLa cells.Conclusions β-TrCP is a novel TRF1 interacting protein; β-TrCP can interact with TRF1 in vivo and in vitro; β-TrCP and TRF1 have colocalization on cells and may play important roles in the functional regulation of cell cycle.