运用双杂交系统探寻与内皮素A型受体相互作用的蛋白质  

Screening proteins interacted with endothelin type A receptor by yeast two hybrid system

作　　者：文立民[1] 张兆山[1] 叶棋浓[1] 李淑琴[1] 曹勇[1] 黄翠芬[1] 

机构地区：[1]北京生物工程研究所

出　　处：《军事医学科学院院刊》1999年第1期19-22,共4页Bulletin of the Academy of Military Medical Sciences

基　　金：国家教育部博士后基金

摘　　要：目的：运用酵母双杂交系统寻找与内皮素Ａ型受体（ｅｎｄｏｔｈｅｌｉｎｔｙｐｅＡｒｅｃｅｐｔｏｒ，ＥＴＡＲ）相互作用的蛋白质，为探讨心血管等疾病的致病机理及采取相应的治疗策略提供理论依据。方法：首先将ＥＴＡＲ胞内区ｃＤＮＡ片段克隆入ｐＧＢＴ９质粒载体中，得到的重组质粒ｐＧＢＴ９－ＥＴＡＲ转化酵母菌ＨＦ７Ｃ，然后将鼠胚胎文库质粒转化到ＨＦ７Ｃ（ｐＧＢＴ９－ＥＴＡＲ）中，用Ｔｒｐ－Ｌｅｕ－Ｈｉｓ－营养缺陷型培养基初步筛选文库中插入ｃＤＮＡ所编码的序列与ＥＴＡＲ胞内区相互作用的菌落，再进一步检测β－半乳糖苷酶活性。结果：共得到数个β－半乳糖苷酶活性为阳性的酵母菌落。提取阳性酵母菌落的质粒，电击转化Ｅ．ｃｏｌｉＨＢ１０１（Ｌｅｕ－），用Ｌｅｕ－培养基筛选只含有文库质粒的菌落。提取质粒ＤＮＡ并测序。结论：文库质粒中插入的ｃＤＮＡ经序列分析和Ｇｅｎｂａｎｋ检索表明，该ｃＤＮＡ编码为与发育相关的蛋白质ｔｅｘ２６１。Objective: To screen the proteins interacting with cytosolic domain of endothelin type A receptor (ET AR) by yeast two hybrid system for studying intracellular signal transduction of endothelin. Methods: The cytosolic cDNA fragment of ET AR was obtained by PCR method. The cDNA fragment was then recombined into the bait vector pGBT9 in correct direction to construct bait fusion expressing vector pGBT9 ET AR. The recombinant pGBT9 ET AR was transformed into yeast host strain HF7C. By electroporation , mouse embryo cDNA library which constructed with library vector pGAD10 was transformed into HF7C containing pGBT9 ET AR. Results: Several His +Lacz + strong positive clones were selected after growing on SD Trp -Leu -His - media and β galactosidase activity assay was done. The library plasmids isolated from the positive clones were characterized by restriction enzymes cutting and DNA sequencing. The results of Genbank sequence similarity searching showed that 5′ region of cDNA fragment of two positive clones may have high sequence similarity with an mRNA from mouse muscle. Conclusion: The cDNA encodes a protein tex261 which interrelates with the development.

关 键 词：双杂交系统 内皮素A型受体 蛋白质 心血管疾病 

分 类 号：R540.2[医药卫生—心血管疾病]