酵母单杂交筛选实验中的关键问题  

Some keys of screening cDNA library with yeast one hybrid system

作　　者：牛永东[1] 许丽艳[2] 韩溟[1] 方王楷[1] 沈忠英[2] 李恩民[1] 

机构地区：[1]汕头大学医学院生化与分子生物学教研室,广东汕头515041 [2]汕头大学医学院肿瘤病理研究室

出　　处：《中国实验诊断学》2006年第7期715-717,共3页Chinese Journal of Laboratory Diagnosis

基　　金：国家自然科学基金面上项目(No.30170428;No.30370641;No.30570849);广东省自然科学基金重点项目(No.37788;No.05104541);中华人民共和国教育部博士学科科研基金项目(No.20020560002;No.20050560003)

摘　　要：目的酵母单杂交系统是研究生物大分子DNA和蛋白质间相互作用的有效手段之一,酵母单杂交筛库实验是获取可以和诱饵序列相互作用的反式作用因子的关键。方法酵母单杂交诱饵质粒pHISi3NF转化YM4271;通过不同浓度3-AT实验,获得酵母单杂交用宿主细胞YM4271pHISi3NF4;大规模筛选人食管癌细胞cDNA文库。结果经筛库后续验证实验证实得到阳性克隆。结论通过准确确定3-AT的工作浓度,有效地完成了酵母单杂交的大规模筛选人食管癌细胞cDNA文库的实验。Objective Screening the cDNA library is the key of the yeast one hybrid technique.Methods Screening the cDNA library of esophageal carcinoma cell line SHEEC with the cell strain YM4271 pHISi3NF4 by LiAc/PEG method in large scale, which is the product of the constructed bait plasmid pHISi3NF4 transforms YM4271 strain, Further study were carried on by the 3-AT higher inhibition test and changed report gene tested test.Results 30 positive elones by 3-AT higher inhibition test and restriction endo-nucleases digested tests. Conclusion The cDNA library of yeast one hybrid system had been screened successfully in large scale by confirming the matched concentration of 3-AT.

关 键 词：酵母单杂交 阳性克隆 CDNA文库 

分 类 号：Q78[生物学—分子生物学]