神经特异性转录因子DAT1酵母双杂交诱饵载体的构建  

Construction of bait plasmid containing neuronal specific transcription factor DAT1 in yeast two-hybrid system

作　　者：惠玲[1] 蔡文琴[2] 王小平[1] 

机构地区：[1]兰州军区兰州总医院医学科学实验中心,甘肃兰州730050 [2]第三军医大学神经生物教研室

出　　处：《西北国防医学杂志》2005年第4期244-246,共3页Medical Journal of National Defending Forces in Northwest China

基　　金：国家自然科学青年基金资助项目(30400125)

摘　　要：目的:用神经特异性转录因子DAT1构建酵母双杂交系统中的诱饵载体。方法:根据GenBank中提供的DAT1序列设计引物,以小鼠脑组织cDNA文库为模板,PCR扩增DAT1,并与pLexA载体连接,测序鉴定。用醋酸锂法转化酵母菌EGY48,在选择性培养基上观察pLexA-DAT1在EGY48中的表达。结果:PCR扩增出的DNA片段约490bp,大小正确,构建的融合表达载体pLexA-DAT1,测序结果表明序列完全正确,融合区域的读码框正确。转化了pLexA-DAT1的酵母菌EGY48在加有X-Gal的SD/Gal/Raf/-His/-Ura营养缺陷型诱导平板上菌落不变蓝,证明LexA-DAT1融合蛋白本身不具有激活p8op-LacZ报告基因的能力,可以用作进一步的实验。结论:获得了在酵母细胞中正确表达且未自主激活报告基因的融合表达载体pLexA-DAT1,可作为酵母双杂交系统中的“诱饵”。Objective ：To construct a bait vector containing neuronal specific transcription factor DAT1 in yeast two hybrid system in order to screen its interaction proteins. Methods： PCR was used to amplify DAT1 from the adult brain cDNA library with the primers designed in accordance with the DAT1 sequence in Genbank . The product was inserted into pLexA vector （ named as pLexA - DAT1 ）. After confirmation with sequence analysis, the plasmid was transformed into the yeast cell EGY48, and the transformants were selected on selective plates. Results： The amplified product of 490 bp was inserted into pLexA vector and the sequence analysis revealed that the sequence was correctly inserted into pLexA with a right reading frame. EGY48 [ pLexA - DAT1] grew on selective plate SD/Gal/Raf/- His/- Ura with X - Gal and was not blue. Conclusion ： The bait plasmid of pLexA - DAT1 expressed DAT1 correctly , and could not activate the transcription of reporter gene alone in yeast two hybrid system.

关 键 词：神经特异性转录因子 酵母双杂交 诱饵质粒 

分 类 号：R346[医药卫生—基础医学]