摘要
目的构建Ku70蛋白酵母双杂交诱饵载体,为研究Ku70及其相互作用蛋白在乳腺癌中的作用机制奠定基础。方法采用反转录聚合酶链反应(RT-PCR)法从人宫颈癌HeLa细胞株扩增出Ku70 cDNA全长片段,用SalⅠ和EcoRⅠ双酶切Ku70 cDNA片段后定向克隆到真核细胞表达载体pGBKT7,获得诱饵质粒pGBKT7-Ku70,经限制性内切酶酶切和测序鉴定后转化到酵母菌株AH109,在营养缺陷培养基中观察pGBKT7-Ku70的自激活作用,同时利用蛋白质免疫印迹检测诱饵蛋白Ku70的表达。结果 Ku70 cDNA正确克隆到载体pGBKT7,转化到酵母菌株AH109中的诱饵载体pGBKT7-Ku70经表型筛选证实无自激活作用,蛋白质免疫印迹检测显示pGBKT7-Ku70在酵母细胞中稳定表达诱饵蛋白Ku70。结论成功构建了Ku70蛋白酵母双杂交诱饵载体,并且在酵母菌株AH109中稳定表达。
Objective To construct a bait vector for Ku70 yeast two-hybrid as a basis for further study of the molecular mechanism of Ku70 role in breast cancer. Methods Reverse transcription polymerase chain reaction（RT-PCR） was used to amplify KuTO eDNA full fragment length from human cervical carcinoma HeLa cells,Sal I and EcoR [ were then used to double digest Ku70 eDNA and directional cloned to eukary- otic expression vector pGBKT7 to get the bait plasmid pGBKTT-Ku70 ,which was then transformed into yeast strain AH109 after restriction enzyme digestion and DNA sequencing, and pGBKT7-Ku70 self-activation was observed in nutritional-deficiency medium, and Western-blot was used to test KuT0 expression. Results KuT0 gene was found in the reconstructed plasmid pGBKTT-Ku70 by sequencing; yeast two-hybrid tests showed that yeast cell AH109 transfected with pGBKTT-KuT0 had no autonomous activation, the expression of bait protein KuTO was confirmed by Western-blot. Conclusion KuT0 yeast two-hybrid bait vector is suc- cessfully contructed,which is stably expressed in geast strain AHI09.
出处
《医学综述》
2013年第22期4168-4171,共4页
Medical Recapitulate
