Expression of Bit1 gene in a yeast two-hybrid system and reporter gene activation assay

AIM: To construct a bait vector with Bit1 gene in yeast two-hybrid system GAL4, and assay whether Bit1 gene expression product affects the growth of host yeast cells and activates the reporter genes in the yeast two-hybrid system. METHODS: The Bit1 gene fragments were amplified by RT-PCR from ovarian cell Caov-3, and cloned into pUC19 for DNA sequencing. After verified by DNA sequencing, they were subcloned into the bait vector pGBKT7 of yeast two-hybrid system GAL4, and the recombinant plasmids were subsequently transferred into yeast cell AH109, and its expression product was tested whether it could activate the reporter genes in the yeast two-hybrid system. RESULTS: The Bit1 gene fragments were successfully amplified, and their expression product showed to be nontoxic to AH109 cells, and did not activate the reporter genes of the GAL4 system. CONCLUSION: Yeast two-hybrid GAL4 system can be utilized to study Bit1-interacting protein.