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

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.