组蛋白酵母双杂交诱饵载体的构建及其自激活作用检测

Construction and Characterization of Yeast Two-Hybrid Bait Vec tors for Histone

目的:构建组蛋白H2A、H2B、H3、H4的酵母双杂交诱饵载体,检测其在酵母细胞中的表达和自激活活性,为应用酵母双杂交系统筛选与组蛋白相互作用的蛋白奠定基础。方法:扩增H2A、H2B、H3、H4蛋白基因编码区cDNA序列,克隆至酵母表达载体pGBK-T7中,再将其转化至酵母细胞AH109中,提取酵母总蛋白,检测各种组蛋白的表达,并检测表达的组蛋白在酵母细胞中对报告基因有无自激活作用。结果:将H2A、H2B、H3、H4基因的cDNA克隆至酵母表达载体pGBK-T7中,其中组蛋白H4得到正确表达,且对报告基因LacZ、HIS3、Ade无激活作用。结论:可以利用酵母双杂交系统筛选与H4相互作用的蛋白质。

Objective： To construct the yeast two-hybrid bait expression plasmids of histone H2A, H2B, H3 and H4, and to detect their protein expression and autonomous activation activity in yeast cells. Methods： The coding regions of H2A, H2B, H3 and H4 were amplified and cloned into the pGBK-T7 yeast two-hybrid vector, and were transformed into yeast AH109 cells. The total yeast protein was then extracted and the expression of the above bistone proteins were detected and their autonomous activation activities were examined. Results： The cod- ing regions of H2A, H2B, H3 and H4 were cloned into the pGBK-T7 vector successfully and histone H4 got ex- pressed and showed no autonomous activation to the reporter gene LacZ, HIS3 and Ade. Conclusion： The yeast two-hybrid system can be applied to screen the interacting proteins of histone H4.