应用酵母双杂交方法筛选LRP16相互作用蛋白

目的:应用酵母双杂交技术筛选人乳腺癌细胞MCF-7中可与LRP16相互作用的蛋白。方法:将诱饵质粒pGBKT7-LRP16、MCF-7双链cDNA文库片段及线性化质粒pGADT7-Rec共转化AH109酵母菌,营养缺陷型培养基(SD/-Leu/-Trp/-His/-Ade)筛选阳性菌落。提取阳性克隆质粒进行PCR,将PCR产物测序,并将酵母质粒转化感受态大肠杆菌Top10获得与诱饵质粒有相互作用的单个文库质粒,测序后回复验证其在酵母中的相互作用。结果:获得8个与LRP16相互作用的候选蛋白,选取4个在酵母中进行回复验证,其中有3个可生长出阳性克隆。结论:应用酵母双杂交技术筛选出一族功能基本明确的可与LRP16相互作用的候选蛋白,为研究LRP16的病理生理功能和分子机制奠定了基础。

Screening of proteins interacting with LRP16 by yeast two-hybrid

Objective:To screen LRP16 interaction proteins from cDNA library of MCF-7 human breast cancer cell line by yeast two-hybrid.Methods: The library was constructed and screened by cotransformation of the double strand cDNA and the linearized pGADT7-Rec with the bait into the AH109 yeast strain.The inserted fragments of identified positive clones were amplified by PCR and sequenced.And library plasmids of positive clones were obtained from competent bacterium coli Top10 transformated with yeast plasmid.Following,the interactions of LRP16 and positive plasmid were tested in yeast cells again.Results:There were 8 different candidate proteins obtaind by Blast analysis in NCBI.Four putative positive library plasmids were further tested interaction in yeast by transformation and three could group yeast clones.Conclusions:We obtain a novel class of LRP16 interacting proteins by yeast two-hybrid system.These results provide essential clues for further investigation of the functional regulation of LRP16 and identification.