LIM结构域蛋白FHL1C与GNB2不存在相互作用  

Study on the interaction between LIM domain protein FHL1C and GNB2

作　　者：刘娟[1] 赵俊龙[2] 王媛媛[2] 韩骅[2] 秦鸿雁[2] 张丙芳[1] 

机构地区：[1]第四军医大学西京医院老年病科,西安710032 [2]第四军医大学医学遗传学与发育生物学教研室肿瘤生物学国家重点实验室

出　　处：《山西医科大学学报》2015年第11期1068-1073,共6页Journal of Shanxi Medical University

基　　金：国家自然科学基金重点资助项目(31130019);国家自然科学基金面上资助项目(31371474)

摘　　要：目的通过哺乳动物双杂交及免疫共沉淀技术验证经质谱技术获得的LIM结构域蛋白FHL1C相互作用候选分子GNB2与FHL1C之间是否存在相互作用,从而丰富FHL1C调控Notch信号通路的机制。方法分别构建融合质粒p CMVGAL4-DBD-FHL1C和p CMV-VP16-GNB2,并将其共转染He La细胞,通过哺乳动物双杂交实验验证两者在细胞内是否具有相互作用;构建带Flag标签的GNB2融合蛋白的重组载体p CMV-Flag-GNB2,酶切鉴定正确后,与带myc标签的FHL1C融合蛋白的重组真核表达载体p CMV-myc-FHL1C共转染He La细胞,利用免疫共沉淀技术检测GNB2与FHL1C之间是否存在生理上的相互作用。结果哺乳动物双杂交实验和免疫共沉淀实验均显示:FHL1C与GNB2之间不存在生理上的相互作用。结论成功构建了实验所需的GNB2的融合蛋白真核表达重组载体,利用哺乳动物双杂交实验及免疫共沉淀技术证实FHL1C与GNB2之间不存在相互作用。Objective To determine the interaction of FHL1 C with GNB2 by mammalian two-hybrid and co-immunoprecipitation,which will further reveal the regulatory mechanisms of FHL1 C in Notch signaling pathway. Methods GAL4-DBD-FHL1 C fusion plasmid p CMV-GAL4-DBD-FHL1 C and VP16-GNB2 fusion plasmid p CMV-VP16-GNB2 were constructed and co-transfected into He La cells,and then the interaction of FHL1 C with GNB2 was detected by mammalian two-hybrid assay. Flag-tagged fusion protein expressing recombinant vector p CMV-Flag-GNB2 was constructed and identified by enzyme digestion. The p CMV-Flag-GNB2 plasmid and myc-tagged fusion protein expressing plasmid p CMV-myc-FHL1 C were co-transfected into He La cells,and then the physical interaction between FHL1 C and GNB2 was detected by co-immunoprecipitation. Results The results of mammalian two-hybrid assay and co-immunoprecipitation experiment showed that there was no physical interaction between FHL1 C and GNB2. Conclusion GNB2 fusion protein expression vectors for mammalian two-hybrid and co-immunoprecipitation experiments have been successfully constructed. The interaction between FHL1 C and GNB2 does not exist.

关 键 词：LIM结构域 FHL1C GNB2 哺乳动物双杂交 免疫共沉淀 

分 类 号：R392.11[医药卫生—免疫学]