应用酵母双杂交技术筛选转录因子MEOX2的相互作用蛋白

Identify the Transcriptional Factor MEOX2 Interacting Proteins Using the Yeast Two-Hybrid Technique

目的研究同源盒基因Meox2所编码的转录因子MEOX2在心肌细胞内与哪些蛋白质存在相互作用。方法应用酵母双杂交技术对人心脏cDNA文库进行筛选。(1)通过PCR方法扩增编码组氨酸/谷氨酸(H/Q)富含区缺失的MEOX2蛋白质(Meox2ΔHQ)的DNA片断,并克隆进入pGBKT7载体,从而构建“诱饵”质粒pGBKT7Meox2ΔHQ;(2)将“诱饵”质粒pGBKT7Meox2ΔHQ转化酵母菌AH109,然后从被转化的酵母菌中提取蛋白质,应用抗C Myc单克隆抗体进行免疫印迹分析,检测“诱饵”蛋白的表达;(3)将被“诱饵"质粒转化的AH109分别涂布于SD/Trp、SD/Trp/His、SD/Trp/Ade和SD/Trp/XαGAL平板上,以观察“诱饵”蛋白自我激活报告基因与否;(4)将已被“诱饵”质粒转化的AH109与人心脏cDNA文库进行杂交,筛选阳性克隆,分离阳性克隆中的cDNA,并测序。结果Meox2ΔHQ与pGBKT7中的Gal4DNA结合域及C myc在同一读框,并稳定表达融合蛋白“Gal4DNA结合域C myc Meox2ΔHQ”,“诱饵”蛋白不会自我激活报告基因,筛选得到11个准阳性克隆。结论同源盒基因Meox2所编码的转录因子MEOX2可能与心肌细胞中的多种蛋白质存在相互作用,并参与这些蛋白质表达的调控。

Objective To identify the proteins in the cardiac myocyte which interact with the transcriptional factor MEOX2. Methods Screen the human heart cDNA library using the yeast two hybrid technique. （ 1 ） A cassette encoding the MEOX2 protein with its histidine/glutamine （H/Q） rich domain deleted （Meox2△HQ） was PCR amplified and then was cloned into the pGBKT7 to construct the ＂bait＂ plasmid; （2） The protein was extracted from the AH109 transformed with the pGBKTT-Meox2△HQ, and then the Western blot was performed to detect the expression of the fusion protein using the anti-Myc antibody ; （ 3 ） The AH109 transformed with pGBKT7-Meox2AHQ was plated on the different synthetic dropout （SD） plates, SD/-Trp, SD/-Trp/-His, SD/-Trp/-Ade and SD/-Trp/X-α-GAL, to determine whether the baits themselves auto-activated the reporter genes; （4） The AH109 transformed with the pGBKT7-Meox2△HQ mated with the human heart cDNA library, and the positive clones were sequenced. Results The Meox2△HQ was in the same frame with the Gal4 DNA binding domain, and the ＂bait＂ protein was expressed stably. The ＂bait＂ protein couldn＇ t auto-activated the reporter genes. After screening a human heart cDNA Library, eleven putative interacting proteins were identified. Conclusion The transcriptional factor MEOX2 encoded by the homeobox gene Meox2 probably interacts with multiple proteins in cardiac myocyte, and participates in the expression regulation of these proteins.