酵母双杂交诱饵蛋白Rb融合质粒构建及鉴定

Construction and confirmation of the fused plasmid with Rb bait gene in yeast two-hybrid system

目的 为筛选和克隆与口腔鳞癌增殖相关的新基因 ,构建酵母双杂交系统用诱饵蛋白融合质粒。方法 从pGBT9 pRb中酶切、电泳鉴定、回收Rb基因 ,将其连接至酵母双杂交系统诱饵蛋白质粒载体 pGBKT7中 ,构建重组质粒pGBKT7 pRb ,并经鉴定、测序、体外翻译、转录等方法验证后 ,将重组质粒转化酵母Y187,测定其在Y187中的自激活现象及毒性。然后 ,提取转化后的酵母总蛋白 ,经Westernblot检测该重组质粒在Y187内的表达情况 ,以鉴定其作为诱饵蛋白的可行性。结果Rb基因经酶切、电泳后 ,大小正确 ,割胶回收 ,与质粒 pGBKT72 4h连接 ,转化大肠杆菌DH5α ,挑选重组质粒 ,测序结果与Genbank中Rb序列比对完全正确。重组质粒pGBKT7 pRb双酶切鉴定、体外翻译、转录试验 ,Westernblot等方法均证实重组质粒中的Rb基因能够正确合成Rb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 pRb ,能够在Y187内正确表达 ,可作为酵母双杂交系统用诱饵蛋白。

Objective To screen and clone the noval genes related to cellular proliferation of oral squamous cell carcinoma. Methods We selected the Rb gene as the bait protein gene to construct the fusion bait plasmid of yeast two-hybrid. The whole code sequence of Rb gene was acquired by digestion with restricted enzyme EcoRI and BamH1 and reclaimed from its original vector pGBT9-pRb. After being confirmed by electrophoresis,the Rb gene was cloned into the MCS of the plasmid pGBKT7 to construct a recombined plasmid pGBKT7-pRb and the sequence of the recombined plasmid was detected in company. According to the protocol of yeast two hybrid system III,the competent Y187 yeast was prepared,and transformed with recombined plasmid pGBKT7-pRb. Following that,the toxicity and transcriptional activation of this recombined plasmid pGBKT7-pRb in Y187 yeast were tested. Results The sequence of the recombined plasmid was correct compared with the sequence provided in Genbank. The protein could be correctly synthesized in vitro,and no self-activating transcriptional activation and toxicity was observed in Y187 yeast. Conclusions The construction of the recombined plasmid was capable to be used as the fusion bait plasmid in yeast two-hybrid system III,and the recombined Rb-protein could be used as the bait protein successfully.