Objective;To construct a bait vector containing human cide -3 gene in yeast two -hybrid system in order to screen the human liver cDNA library. Methods; The fragment including all exons of cide -3 gene was amplified by RT-PCR and was inserted into the multiple cloning sites of the shuttle vector pGBKT7. After confirmation with restricted endonuclease digestion and sequence analysis,by using PEG/LiAC method,the plasmid pGBKT7 - cide -3 was transformed into yeast AH109. The plasmid and protein isolated from the positive yeast AH109 clone was tested its expression,toxicity and transcriptional activation. Results:The human cide -3 cDNA was amplified. The restrictive endonuclease digestion and DNA sequencing proved that the plasmid pGBKT7 - cide - 3 was obtained. The yeast AH109 clone transformed with pGBKT7 - cide - 3 was screened out by the SD/ - Trp nutritional media. Sequence analysis revealed that the fragment was correctly inserted into pGBKT7 with a right reading frame and its expression in yeast was verified. Conclusion; The bait plasmid pGBKT7 -cide -3 constructed expresses correctly,and can not activate the transcription of reporter gene alone in yeast two - hybrid system.%目的:构建并转化酵母双杂交体系中“诱饵”载体pGBKT7-cide-3以便用于人肝cDNA文库的筛选.方法:用RT-PCR方法从人肝细胞株QZG中扩增cide-3基因全外显子片段,并定向克隆入细菌-酵母穿梭表达质粒pGBKT7中；用PEG/LiAC法将pGBKT7-cide-3转化感受态酵母菌AH109.提取转化酵母菌的质粒DNA和总蛋白进行鉴定.同时检测表达产物对报告基因的激活作用及对酵母株AH109的毒性.结果:成功扩增出人cide-3 cDNA片段,限制性内切酶酶切和DNA测序证实获得正确的重组质粒pGBKT7-cide-3；获得可在色氨酸缺陷培养基(SD/-Trp)上生长含pGBKT7-cide-3的酵母菌克隆.鉴定表明pG-BKT7-cide-3已转化入酵母菌AH109,无自主激活报告基因的能力及对酵母的生长无毒性.结论:酵母双杂交“诱饵”载体pGBKT7-cide-3构建获得成功,可应用于酵母双杂交体系.
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