Expression of Bit1 gene in a yeast twoª²hybrid system and reporter gene activation assay

    HUA Wei1, ZHANG Wei2, L¦a Haiª²Peng3, XIN Xiaoª²Yan1

    1Department of Obstetrics and Gynecology, Xijing Hospital, 2Department of Biochemistry and Molecular Biology, School of Basic Medicine, 3Department of Endodontics, School of Stomaª²tology, Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To construct a bait vector with Bit1 gene in yeast twoª²hybrid system GAL4, and assay whether Bit1 gene expression product affects the growth of host yeast cells and activates the reporter genes in the yeast twoª²hybrid system. METHODS: The Bit1 gene fragments were amplified by RTª²PCR from ovarian cell Caovª²3, and cloned into pUC19 for DNA sequencing. After verified by DNA sequencing, they were subcloned into the bait vector pGBKT7 of yeast twoª²hybrid 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 twoª²hybrid 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 twoª²hybrid GAL4 system can be utilized to study Bit1ª²interacting protein. ......