人FAM92A1基因诱饵表达质粒的构建与鉴定

Construction and Identification of Bait Expression Plasmid of Human FAM92A1

目的 构建人FAM92A1基因（hFAM92A1）的诱饵表达质粒pGBKT7-hFAM92A1并检测其蛋白表达、毒性和自激活作用.方法 PCR扩增hFAM92A1的基因编码序列并克隆入诱饵表达载体pGBKT7中,酶切和测序鉴定后,转化到酵母AHl09细胞中,Western印迹检测诱饵蛋白表达情况,同时检测诱饵蛋白的毒性和自激活作用.结果 成功构建FAM92A1基因的诱饵表达质粒pGBKT7-hFAM92A1,测序结果正确.Western印迹实验证实酵母细胞高表达诱饵蛋白hFAM92A1,诱饵蛋白没有自激活作用.结论 构建的诱饵表达质粒pGBKT7-hFAM92A1可用于下一步酵母双杂交系统实验,为进一步研究hFAM92A1功能奠定了基础.

Objective To construct the bait expression plasmid pGBKT7-hFAM92A1 and detect its protein expression, toxicity and serf-activation. Methods The sequence of human FAM92A1 was amplified by PCR, and then was cloned into pGBKTT. After verified by cutting and sequencing, the constructed pGBKTT-hFAM92A1 plasmid was transformed into yeast cells AH109, and the expres- sion of the bait protein Was analyzed by Western Blot. Toxicity and serf-activation of the bait protein were detected. Results The sequence of human FAM92A1 was amplified and cloned into pGBKT7 successfully. Transformation of the bait expression tivating effect. Expression of the bait protein was plasmid into yeast cells AH109 did not have self-ac- confirmed by Western Blot, showing no toxicity to yeast AH109 cell. Conclusion The construct of pGBKT7-hFAM92A1 can correctly encode human hFAM92A1 protein in yeast cells, and can be used as a bait in yeast two-hybrid system.