分化抑制因子ID1’基因酵母双杂交诱饵载体的构建及自激活作用的检测

Construction of ID1’ bait vector and detection of its self-activating effect in yeast two hybrid system

目的 :构建酵母双杂交用分化抑制因子ID1’基因的诱饵载体 ,并检测其自激活作用。方法 :PCR扩增ID1’基因全长 ,酶切后与诱饵载体pHybLex/Zeo连接形成重组诱饵载体 ,电穿孔方法转化到酵母细胞EGY48中 ,β -半乳糖苷酶滤膜分析检测重组诱饵载体对报告基因LacZ的自激活情况。结果 :扩增出ID1’基因全长 ,成功构建了重组诱饵载体pHybLex/Zeo -ID1’ ,经酶切和测序鉴定正确 ;重组诱饵载体无自发激活报告基因功能。结论 :重组诱饵载体构建成功 ,对报告基因无自激活作用 。

Objective:To construct the bait vector of ID1’ gene in yeast two hybrid system and examine whether the bait vector of ID1’has self-activating effect.Methods:The full length of ID1’ gene was amplified by PCR. The amplified fragment of ID1’ was ligased with the vector pHybLex/Zeo to construct recombinant plasmid phyblex/Zeo-ID1’.The recombinant plasmid was transfected into yeast strain EGY48/pSH18-34 by electrotransfection protocol, subsequently the self-activating effect was observed by β-galactosidase filter assay.Results:ID1’ gene was amplified and ligased with pHybLex/Zeo. Digested with endonuclease and sequenced, the recombinant plasmid of pHyblex/Zeo-ID1’ was constructed correctly. Experimental result of β-galactosidase filter assay suggested that the recombinant plasmid didn’t have self-activating effect.Conclusion:The recombinant plasmid of pHybLex/Zeo-ID1’ was constructed successfully and it didn’t display self-activating effect . This step paves the way for further screening cDNA library.