带核定位信号的维甲酸受体α与Ubiquilin 1蛋白相互作用的验证(英文)  

Interaction between nuclear localization signal-retinoic acid receptor α and Ubiquilin 1

作　　者：朱丹[1] 王翀 刘北忠[1,2] 吴燕[1] 钟梁[1] 王春光[1] 

机构地区：[1]重庆医科大学医学检验系临床检验诊断学教育部重点实验室,重庆400016 [2]重庆医科大学附属永川医院中心实验室,重庆402160

出　　处：《中南大学学报（医学版）》2010年第7期649-654,共6页Journal of Central South University ：Medical Science

基　　金：supported by National Natural Science Foundation of China (30300449);the Traditional Chinese Medicine Foundation of State Administration (02-03ZP52 );the Fund from Chongqing Medical University (XBYB2007104,XBYB2007108)

摘　　要：目的:验证带有核定位信号的维甲酸受体α(nuclear localization signal-retinoic acid receptorα,NLS-RARα)与Ubiquilin 1(UBQLN1)蛋白之间的相互作用。方法:将表达NLS-RARα诱饵蛋白和UBQLN1靶蛋白的两种重组表达质粒pGBKT7-NLS-RARα及pACT2-UBQLN1共转化AH109酵母菌,通过酵母双杂交技术验证两者在活细胞内的相互作用;构建真核细胞表达载体pCMV-HA-NLS-RARα和pCMV-Myc-UBQLN1,经酶切鉴定后,共转染HEK293细胞,利用抗HA多克隆抗体沉淀,抗Myc单克隆抗体检测验证他们之间的相互作用。结果:pGBKT7-NLS-RARα及pACT2-UBQLN1质粒共转化AH109酵母菌后,可见蓝色阳性克隆;构建的重组表达载体pCMV-HA-NLS-RARα和pCMV-Myc-UBQLN1经酶切鉴定成功,转染HEK293细胞后,用免疫共沉淀技术检测到Myc-UBQLN1蛋白的表达。结论:酵母双杂交和免疫共沉淀技术可验证NLS-RARα与UBQLN1之间存在相互作用。Objective To identify the interaction between nuclear localization signal-retinoic acid receptor α（NLS-RARα） and Ubiquilin 1（UBQLN1）.Methods The recombination expression plasmids pGBKT7-NLS-RAR and pACT2-UBQLN1,which expressed bait-protein NLS-RARα and target protein UBQLN1 respectively,were cotransformated into yeast AH109.The interaction of the expression plasmids in the living cells was investigated by yeast two-hybrid assay.HA-tagged fusion protein（pCMV-HA-NLS-RARα） expression vector and Myc-tagged fusion protein（pCMV-Myc-JTV1） expression vector were constructed,identified,and cotransfected respectively into human embryo kidney 293 cells（HEK293）.The interaction was detected by co-immunoprecipitation.Results Blue clones were found on yeast AH109 plate cotransformated with pGBKT7-NLS-RARα and pACT2-UBQLN1.Double restriction enzyme digestion showed that pCMV-HA-NLS-RARα and pCMV-Myc-JTV1 were successfully constructed.Then HA-NLS-RAR protein was immunoprecipitated by anti-HA polyclonal antibody and the expression of Myc-UBQLN1 was tested by Western blot with anti-Myc monoclonal antibody from immunoprecipitated complex.Conclusion The interaction between NLS-RARα and UBQLN1 can be verified by yeast two-hybrid assay and co-immunoprecipitation.

关 键 词：酵母双杂交 免疫共沉淀 蛋白质相互作用 

分 类 号：Q75[生物学—分子生物学]