中文摘要：

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。

英文摘要：

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 at1 exons of cide - 3 gene was ampli- fied 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 - ci- de- 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 re- strictive endonuelease digestion and DNA sequencing proved that the plasmid pGBKT7 - cide - 3 was obtained. The yeast AH109 clone transformed with pGBKT7 - eide - 3 was screened out by the SD/- Trp nutritional media. Se- quence analysis revealed that the fragment was correctly inserted into pGBKT7 with a right reading frame and its ex- pression 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.