靶向敲减Ras癌蛋白融合型泛素连接酶E3的构建  

Construction of Chimeric E3s Expression Plasimds Targeting Oncoprotein Ras

作　　者：马怡晖[1,2] 张强[1] 谷雨妹[1] 卢朝辉[1] 陈杰[1] 

机构地区：[1]中国医学科学院北京协和医学院北京协和医院病理科,北京100730 [2]郑州大学第一附属医院病理科,郑州450052

出　　处：《中国医学科学院学报》2012年第4期313-318,共6页Acta Academiae Medicinae Sinicae

基　　金：国家自然科学基金(30270599、3047197、30973470、81172334);卫生部卫生行业科研专项项目(200802011);教育部高等学校博士学科点专项科研基金(20060023013)~~

摘　　要：目的应用蛋白质敲减技术原理,构建理论上能够靶向敲减Ras癌蛋白的融合型泛素连接酶E3的真核表达载体。方法选取Raf-1、PI3K、RalGDS中能够与Ras蛋白相互作用的结合结构域和具有泛素连接酶活性的F-Box及U-Box作为功能结构域,采用分子克隆技术依次将其连入pcDNA3.1中构建融合蛋白表达载体,双酶切、PCR和测序技术检测连入核苷酸片段的正确性,Western blot检测融合蛋白表达载体在真核细胞内表达的正确性和作用有效性。结果成功获得6个融合蛋白E3表达载体,5个能够在真核细胞内有效表达,其中(RBD+CRD)Raf-1-U-Box-pcDNA3.1能够敲减人胰腺癌细胞系PANC-1细胞中Ras蛋白。结论成功构建能够靶向敲减Ras癌蛋白的融合蛋白表达载体,为下一步应用蛋白质敲减技术奠定了实验基础。ABSTRACT： Objective To construct certain chimeric E3s expression plasmids targetting oncoprotein Ras by harnessing the theory of protein knockdown. Methods We chose the binding domain of Raf-1, PI3K, Ral- GDS, and the function domain of F-Box as well as the U-Box to construct the plasmids. Then used the double en- zyme, PCR, and sequence to test the validity and integrity of the cloned nucleotide fragments. The expression ef- ficiency of the plasmids in eukaryotic cells was detected by Western blot analysis. Results Five of 6 plasmids in this study expressed the corresponding fusion proteins in HEK293T ceils, and （ RBD ＋ CRD） Raf-l_ U-Box-pcD- NA3.1 can knocked down the protein level of Ras in PANC-1 cells. Conclusions We successfully constructed the chimeric E3 expression plasmids, which provides a solid basis for further research on protein knockdown.

关 键 词：泛素 蛋白酶体 泛素连接酶 Ras癌蛋白 

分 类 号：R446.8[医药卫生—诊断学]