雄激素受体相关蛋白267-α酵母双杂交诱饵重组质粒的构建及转化  

Construction and transformation of yeast bait plasmids carrying the androgen receptor-associated protein 267-α cDNA fragments

作　　者：买铁军[1] 汪欣[2] 张志文[3] 师长进[1] 辛殿旗[1] 郭应禄[1] 

机构地区：[1]北京大学泌尿研究所,100034 [2]北京大学医院普外科 [3]北京大学第一医学部生理教研室

出　　处：《中华实验外科杂志》2003年第9期815-816,共2页Chinese Journal of Experimental Surgery

摘　　要：目的　构建和转化雄激素受体相关蛋白 2 67 α(ARA2 67 α)的酵母诱饵重组质粒 ,为通过酵母双杂交研究ARA2 67 α的功能奠定基础。方法　用PCR扩增ARA2 67 α全长cDNA中编码PWWP和PHD SET蛋白结构域的两个基因片段 ;将基因片段与 pGBKT7载体定向重组 ;用酶切和测序鉴定重组质粒 ;将核苷酸序列正确的重组质粒转化入AH 10 9酵母菌株。结果　成功构建pGBKT7 PWWP和 pGBKT7 PHDSET重组质粒。分别转化有 2种重组质粒和pGBKT7空载体的3种AH10 9酵母都能在SD/ Trp/X α gal培养基中长成白色菌落 ,但都不能在SD/ His/ Trp/2 .5mmol/L 3 AT/X α gal和SD/ Ade/ Trp/X α gal培养基中生长 ,在SD/ Trp液中培养 16h后 ,A60 0均值分别为 0 .8、0 .9、0 .9。这表明重组质粒表达的融合蛋白没有激活HIS3、ADE2和MEL1酵母报告基因表达的活性 ,也没有酵母毒性作用。Objective To construct and transform yeast bait plasmids carrying the androgen receptor-associated protein 267-α (ARA267-α) cDNA fragments.Methods Fragments of ARA267-αcDNA that respectively code the PWWP and PHD-SET conserved domains were amplified using PCR and directionally ligated to the pGBKT7 vector.Insert-contained plasmids were confirmed by restriction analysis and DNA sequencing and then transformed into AH109 yeast strain.Results pGBKT7-PWWP and pGBKT7-PHDSET recombinant plasmids were successfully constrcted.All three sorts of yeasts respectively transformed recombinant plasmids and empty pGBKT7 vector could grow white colonies on SD/-Trp/ X-α-gal plates and none could survive on SD/-His/-Trp/2.5 mmol/L 3-AT/X-α-gal and SD/-Ade/-Trp/X-α-gal plates.After being cultured in SD/-Trp liquid medium for 16 h the A 600 of them was 0.8,0.9,0.9 respectively.This indicated the fusion proteins expressed by recombinant plasmids did not activate the expression of yeast reporter genes HIS3,ADE2 and MEL1 and had no toxicity to yeast strain.Conclusion The bait plasmids constructed can be used to study the function of ARA267-αin Yeast Two-Hybrid Screen.

关 键 词：雄激素受体 相关蛋白267—α 酵母双杂交技术 诱饵重组质粒 

分 类 号：R346[医药卫生—基础医学]