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目的:构建用于酵母双杂交系统的诱饵载体pDBLeu-Dnd1,并检测其是否有自身激活作用。方法:设计引物引入SalⅠ、NotⅠ酶切位点,使用PCR方法从质粒pcDNA3.1-Dnd1中扩增小鼠Dnd1基因的编码区序列,连至pMD18-T载体。酶切验证及测序正确后,用SalⅠ,NotⅠ内切酶分别消化pMD-T-Dnd1及pDBLeu载体而后构建诱饵载体pDBLeu-Dnd1,并检测其在G IBCO/BRL公司的酵母双杂交系统中的自身激活作用。结果:成功构建诱饵载体pDBLeu-Dnd1,并证明其在该系统中无自身激活作用。结论:pDBLeu-Dnd1可应用该酵母双杂交系统筛选与之相互作用的蛋白。
OBJECTIVE: To construct bait vector pDBLeu-Dnd1 for yeast two-hybrid system and test whether it has self-activation. Methods: Primers were designed to introduce Sal Ⅰ and Not Ⅰ restriction sites. The coding region of mouse Dnd1 gene was amplified from plasmid pcDNA3.1-Dnd1 by PCR and ligated into pMD18-T vector. After digestion and sequencing, the bait vector pDBLeu-Dnd1 was constructed by digesting the pMD-T-Dnd1 and pDBLeu vectors respectively with SalI and NotI endonucleases and detecting their own in the yeast two-hybrid system of GIBCO / BRL Activation. Results: The decoy vector pDBLeu-Dnd1 was successfully constructed and proved to be inactive in this system. Conclusion: pDBLeu-Dnd1 can be used in this yeast two-hybrid system to screen for proteins that interact with it.