Peroxiredoxin Ⅱ酵母双杂交诱饵载体的构建及自激活效应检测  

Construction and Identification of a Yeast Two-hybrid Bait Vector for Screening of Homo Sapiens Peroxiredoxin Ⅱ-binding Protein

作　　者：刘萍[1] 奚玲[1] 陶海鹰[2] 卢运萍[1] 马丁[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院肿瘤生物医学中心,湖北武汉430030 [2]武汉大学人民医院骨科,湖北武汉430060

出　　处：《武汉大学学报（医学版）》2007年第4期413-417,F0002,共6页Medical Journal of Wuhan University

基　　金：国家重点基础研究发展计划资助项目(编号:2002CB513100);国家青年自然科学基金资助项目(编号:30500596)

摘　　要：目的:应用酵母双杂交体系,构建人peroxiredoxinⅡ(PrxⅡ)蛋白酵母双杂交诱饵载体,并检测该载体的表达产物对酵母细胞有无毒性作用及对报告基因有无激活作用。方法:用PCR扩增PrxⅡ基因cDNA中编码完整开放阅读框的基因片段;将该基因片段与pGBKT7载体定向重组;用酶切和测序鉴定重组质粒;用醋酸锂法(Li-Ac)将序列正确的重组质粒pGBKT7-PrxⅡ转化入AH109酵母菌株,在缺陷性培养基上观察pGBKT7-PrxⅡ在AH109中的表达情况,检测诱饵载体有无毒性作用和单倍体及二倍体自激活功能。结果:成功构建pGBKT7-PrxⅡ重组质粒。转化有重组质粒和pGBKT7空载体的酵母菌都能在SD/-Trp/X--αgal平板上长出粉红色菌落,在SD/-His/-Trp/X--αgal,SD/-Ade/-Trp/X--αgal平板上不能生长,两种酵母菌在SD/-Trp液体培养基中培养16 h后,菌液的OD600均值均为0.9,说明AH109[pGBKT7-PrxⅡ]转化成功,对酵母菌株AH109无毒性且不具自主激活报告基因的功能。结论:成功获得了在酵母细胞中正确表达,并对酵母细胞无毒性且未自主激活报告基因的诱饵表达载体pGBKT7-PrxⅡ,该载体可作为酵母双杂交系统中的“诱饵”,该诱饵载体可应用于酵母双杂交系统3筛选PrxⅡ相互作用蛋白。Objective： To construct and identify a yeast two hybrid system bait vector for screening of homo sapiens Prx Ⅱ binding proteins, and to study its effect on the growth of yeast cells and the activation of reporter genes. Methods： Full fragment of ORF of peroxiredoxin Ⅱ cDNA was amplified using PCR and directly ligated to the pGBKT7 vector. Insert-contained plasmid was confirmed by restriction endonuclease analysis and DNA sequencing. The plasmid was transformed into the yeast cell AH109, and its toxicity and transcriptional activation was tested by both the phenotype assay and the color assay. Results： The fragments of Prx Ⅱ protein were successfully obtained. The recombinant pGBKT7-Prx Ⅲ plasmids and empty pGBKT7 vector could grow white colonies on SD/-Trp/X-α-gal plates and none could survive on SD/-His/-Trp/X-α-gal and SD/- Ade/-Trp/X-α-gal plates. After being cultured in SD/-Trp liquid medium for 16 h, the OD600of them were both 0.9. Conclusion： The bait plasmid pGBKTT-Prx Ⅱ constructed expresses correctly, and can not activate the transcription of reporter gene alone. The yeast two hybrid GAL4 system3 can be utilized to fish Prx Ⅱ region interacting protein.

关 键 词：PEROXIREDOXIN Ⅱ 酵母双杂交系统 基因 克隆 

分 类 号：Q554.6[生物学—生物化学] Q782