一个可能的与人端粒结合因子相互作用蛋白Tara的分离和克隆

Isolation of Tara protein and its gene cloning

目的 :分离和鉴定端粒结合因子 (TRF1)免疫共沉淀蛋白复合物并克隆其基因。方法 :以 TRF1抗体应用免疫共沉淀方法 ,从细胞蛋白抽提物中分离 TRF1蛋白复合物 ,并作蛋白质肽指纹谱鉴定 ;应用温度梯度 PCR,从人 c DNA文库中扩增目的基因 ,并构建 p EGFP- C2真核表达载体 ;核苷酸测序和 Western blot验证目的基因序列和表达载体。结果 :从 TRF1免疫沉淀复合物中分离出了 Tara蛋白 ;人 c DNA文库中 PCR扩增所得产物为 1.7kb,与 Tara基因 CDS序列同源性为 99.9% ;GFP/ Tara融合蛋白约 10 0 k D,Tara在间期 He La细胞中弥散表达于胞浆 ,而有丝分裂细胞则定位于整个细胞。结论 :Tara是一个可能的 TRF1相互作用蛋白 ,其作用机制需进一步实验研究。

Objective: To isolate and identify TRF1 immunoprecipitating protein complex and to clone the candidate gene. Methods: The co immunoprecipitation assay was employed to isolate TRF1 protein complex and the immunoprecipitate was subjected to MALDI TOF mass spectrometry for protein identification.The candidate gene was amplified by temperature gradient PCR from human testis cDNA library and then cloned into pEGFP C2 vector for eukaryotic expression.The amplified gene was verified by direct sequencing and GFP tagged protein was confirmed by immunoblotting. Results: Tara protein with the size of 68kD was identified from the TRF1 precipitate.The candidate gene amplified from cDNA library was about 1.7 kb as expected.Sequencing demonstrated the amplified fragment had 99.9% of homogenesis with Tara CDS sequence (gi:30474869).GFP tagged fusion protein was about 100 kD.Tara was diffusely distributed in cytoplasm at interphase and in whole cells at mitotic phase. Conclusion: Tara might be an interacting protein with TRF1. However,further investigation would be required to confirm if they were bona fide partners. [