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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 new genes related to the proliferation of oral squamous cell carcinoma and construct bait protein fusion plasmids for yeast two-hybrid system. Methods The recombinant plasmid pGBKT7 pRb was digested with pGBT9 pRb and identified by electrophoresis. The recombinant plasmid pGBKT7 pRb was ligated into the bait plasmid pGBKT7 of yeast two-hybrid system. After identification, sequencing, in vitro translation and transcription, The recombinant plasmid was transformed into yeast Y187 and its self-activation in Y187 and its toxicity were tested. Then, the total yeast protein was extracted and the expression of the recombinant plasmid in Y187 was detected by Western blot to identify its feasibility as a bait protein. Results The Rb gene was digested by restriction enzyme and electrophoresed. After being excised, it was recovered by tapping and ligated with plasmid pGBKT72 for 4h. The recombinant plasmid was transformed into E. coli DH5α. The sequencing result was completely correct in comparison with the Rb sequence in Genbank. Recombinant plasmid pGBKT7 pRb double digestion identification, in vitro translation, transcriptional assay, Westernblot and other methods were confirmed Rb gene in the recombinant plasmid can correctly synthesize Rb protein. After transformation of yeast, the self-activation of the recombinant plasmid was ruled out. Conclusion The recombinant plasmid pGBKT7 pRb can be correctly expressed in Y187 and can be used as bait protein in yeast two-hybrid system.