FOXL2酵母双杂交诱饵载体构建及检测  

Construction and Detection of FOXL2 Bait Plasmid of Yeast Two-hybrid System

作　　者：张伟[1] 黄旭东 韩小波[3,1] 任珊珊[1] 王小柯[1] 李艳艳[1] 唐胜建[1] 

机构地区：[1]潍坊医学院整形外科医院,山东潍坊261042 [2]潍坊眼科医院,山东潍坊261041 [3]莒县人民医院骨二科,山东日照276500

出　　处：《生物技术》2011年第3期6-10,共5页Biotechnology

基　　金：国家科学自然基金项目("小睑裂综合征(BPES)致病基因的定位克隆";30772268)资助

摘　　要：目的:用FOXL2编码区片段构建酵母双杂交诱饵质粒并对诱饵质粒进行自激活活性及毒性检测。方法:PCR扩增FOXl2编码区DNA片段,克隆入pGBKT7质粒,转化至DH-5α大量扩增后提取质粒,经PCR、酶切和测序验证,再转化至Y187酵母菌株中用缺陷培养基筛选并进行自激活和毒性检测。结果:获得FOXL2基因1 137bp片段,并成功将人类正常和一突变型FOXL2编码区克隆入pGBKT7中,经检测在SD/-Trp/X-α-Gal单缺培养基平板上无蓝色菌斑出现,在SD/-Ade/-Trp培养基平板无生长。同时在SD/-Trp/Kana(20μg/ml)培养24h后测得菌液OD600≥0.8,说明重组诱质粒无自激活活性和酵母毒性,满足作为诱饵质粒的条件。结论:成功构建了正常及突变型的FOXL2酵母杂交诱饵质粒;为进一步利用酵母双杂交技术研究与FOXL2相互作用的蛋白打下坚实基础。Objective： To construct the bait vector of forkhead box L2 （FOXL2） in yeast two - hybrid system, and test the bait vector for transcriptional activation and toxicity. Method ：The open - reading frame （ORF） of FOXL2 was amplified by PCR, and then was cloned into pGBKT7 - Rec. After being verified by PCR, sequencing and digested by restriction enzymes, it was introduced into the yeast cell Y187. Its transcriptional activation and toxicity effect was tested by auxotrophic selective culture. Result： The 1 137bp open- reading frame of FOXL2 was amplified successfully. The mutational FOXL2 recombinant bait plasmid and nonmutational FOXL2 recombinant bait plasmid were cloned into pGBKT7. Blue plaque was not detected on SD/- Trp/X - ct - Gal. And no colony was grown on SD / - Ade/- Trp. At the same time, OD^o of the culture in SD! - Trp/Kana （20ttg/ml） were all I〉0.8. So the bait plasmid didn＇t have transcriptional activation and did not show toxicity to yeast Y187 cell. Conclusion：The bait vector FOXL2 in yeast two - hybrid system is constructed successfully. These lay a rigid basis for investigating the proteins interacting with FOXL2 by yeast two - hybrid technique.

关 键 词：FOXL2 BPES 酵母双杂交 

分 类 号：Q51[生物学—生物化学]