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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 ,为下一步cDNA文库筛选奠定了基础
OBJECTIVE: To construct bait vector of differentiation inhibitor ID1 ’in yeast two-hybrid system and test its self-activation. Methods: The full - length cDNA of ID1 ’was amplified by PCR and ligated with bait vector pHybLex / Zeo to construct a recombinant bait vector. The recombinant bait vector was transformed into yeast cell line EGY48 by electroporation. The recombinant bait vector was detected by β - galactosidase Reporter LacZ self-activation. Results: The full-length cDNA of ID1 was amplified and the recombinant bait vector pHybLex / Zeo-ID1 was successfully constructed. The recombinant bait vector was identified by restriction enzyme digestion and sequencing. The recombinant bait vector did not spontaneously activate the reporter gene. Conclusion: The recombinant bait vector was successfully constructed and had no self-activation effect on the reporter gene, which laid the foundation for the next screening of cDNA library