用于筛选COPS3相互作用蛋白酵母双杂交诱饵载体pGBKT7-COPS3的构建及鉴定

目的 用COPS3基因片段构建酵母双杂交GAL4系统的诱饵载体pGBKT7-COPS3，并检测其表达产物对酵母细胞有无毒性及对报告基因有无激活作用。方法 运用RT-PCR技术从骨肉瘤细胞系SOSP-9607中扩增出COPS3基因片段，克隆入pUC19质粒，经测序正确后，插入酵母双杂交诱饵载体pGBKT7中，将重组质粒导入酵母菌AH109，检测其表达产物在酵母细胞中对报告基因有无激活作用。结果 成功获得COPS3基因片段，其表达蛋白对酵母菌AH109无毒性，对报告基因亦无激活作用。结论可以利用酵母双杂交GAL4系统来研究与COPS3相互作用蛋白。

Construction and identification of bait vector pGBKT7-COPS3 for screening COPS3 interaction protein

Objective To construct bait vector pGBKTT-COPS3 in yeast two-hybrid system GAIN,and explore whether expression product of COPS3 has toxicity to host yeast cells and activates the reporter genes in the yeast two-hybrid system. Methods The COPS3 gene fragments were amplified by RT-PCR from SOSP-9607 ,and cloned into pUC19 for DNA sequencing. After verified by sequencing,it was subcloned into the bait vector pGBKT7 of yeast two-hybrid system GAIN ,and the recombinant plasmid was trans-ferred into yeast cell AH109. The activation of expression product to the reporter genes was tested in the yeast two-hybrid system. Results The COPS3 gene fragments were successfully amplified, and the expression product had no toxicity to AH109 cells, and could not activate the reporter genes of the GAL4 system. Conclusion Yeast two-hybrid GAL4 system can be utilized to study COPS3 interaction protein.