酵母双杂交诱饵蛋白RGS4融合表达质粒的构建及鉴定

Construction and Verification of Bait Protein RGS4-fusion Expression Plasmid in Yeast Two-hybrid System

目的研究糖皮质激素受体各个结构域与G蛋白信号转导负调节因子4(RGS4)之间是否存在相互作用,构建酵母双杂交系统诱饵蛋白融合质粒。方法采用RT-PCR方法扩增RGS4片断全长,酶切回收,将其连接至酵母双杂交系统诱饵蛋白质粒载体pGBKT7,构建重组质粒pGBKT7-RGS4,并经酶切和测序等方法鉴定后,使用Y187酵母菌检测重组质粒的自激活现象及毒性。然后,提取酵母总蛋白,采用Westernblot方法检测重组质粒在Y187内的表达情况,以鉴定其作为诱饵蛋白的可行性。结果RT-PCR扩增的RGS4基因片断电泳后,大小正确,重组质粒,pGBKT7-RGS4测序结果与GenBank中RGS4基因序列比对完全正确。重组质粒pGBKT7-RGS4双酶切鉴定、体外转录翻译实验,Westernblot等方法均证实重组质粒中的RGS4基因能够正确合成RGS4蛋白。转化酵母后排除重组质粒的自激活作用。结论构建重组质粒pGBKT7-RGS4,能够在Y187内正确表达,可作为酵母双杂交系统诱饵蛋白使用。

Objective To research the interaction between glueocorticosteroid receptor domains and regulator of G protein signaling 4 （RGS4） protein. Methods The RGS4 gene was used as the bait protein gene to construct the fusion bait expression plasmid of yeast two-hybrid. The whole encoding sequence of RGS4 gene was amplified by RT-PCR method. With confirmed by electrophoresis, the RGS4 gene was cloned into the MCS of the plasmid pGBKT7 to form a recombinant plasmid pGBKT7-RGS4 and the sequence of the recombinant plasmid was sequenced. According to the protocol of yeast two-hybrid system gold Ⅲ , the competent Y187 yeast was prepared and transformed with recombinant plasmid pGBKT7-RGS4. Following that, the toxicity and autonomous activation of this recombinant plasmid pGBKT7-RGS4 in Y187 yeast were tested. In the end, we verified the normal expression of the RGS4 fusion protein in vitro by TNT system and in vivo by Weston blot. Results The sequence of the recombinant plasmid was verified to be correct, as compared with the sequence provided by GenBank. The protein could be correctly synthesized in vivo, and no autonomous activation and toxicity was observed in Y187 yeast. Conclusions The recombinant plasmid may be used as the fusion bait plasmid in yeast two-hybrid system Ⅱ , and the recombinant RGS4 fusion protein can be used as the bait protein successfully.