串联亲和纯化技术筛选手足口病病毒ev71 3d蛋白 - 第三军医大学学报.doc

串联亲和纯化技术筛选肠病毒71型 3D聚合酶的相互作用蛋白 江月1,2，丛浩龙2，王健2，李梨1（400016 重庆，重庆医科大学：生物化学与分子生物学教研室1；100101 北京，中国科学院微生物研究所2） [摘要] 目的 利用串联亲和纯化技术筛选与肠病毒71型3D聚合酶相互作用的宿主细胞蛋白。方法 利用RT-PCR方法从EV71BrCr株全基因组中获得3D聚合酶全长基因，构建pcDNA3.0-3D-Flag-HA真核表达载体并转染RD细胞，48h后收集细胞。用Western blot方法检测3D蛋白在RD细胞内的表达情况。串联亲和纯化技术筛选与3D聚合酶相互作用的宿主蛋白并进行质谱分析, 确定与3D聚合酶相互作用的蛋白或多肽。并采用免疫共沉淀实验，对候选蛋白CyclinG1与3D的相互作用进行验证。结果 成功构建了pcDNA3.0-3D-Flag-HA载体，在RD细胞内检测到3D蛋白的表达。经串联亲和纯化和质谱分析得到LATS2、Nek2、APC5、Cyclin-G1、PI3K等一系列参与细胞凋亡及细胞周期调控的蛋白。免疫共沉淀实验证实3D蛋白与CyclinG1的相互作用。结论 3D聚合酶可能与宿主细胞内蛋白相互作用，引起细胞凋亡及细胞周期改变。 [关键词] 肠道病毒71型；3D聚合酶；串联亲和纯化；液相色谱-质谱分析法 [中图法分类号] Q71; Q291; Q51 [文献标识码] A Screening of Host Proteins Interacting with Enterovirus 71 3D Polymerase Using Tandem Affinity Purification Jiang Yue1,2, Cong Haolong2, Wang Jian2, Li Li1 (1Department of Biochemistry and Molecular Biology, Chongqing Medical University, Chongqing, 400016; 2Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China) [Abstract] Objective To screen EV71 3D polymerase-interacting proteins by tandem affinity purification(TAP). Methods The full-length 3D polymerase gene was amplified from the whole genome of EV71 BrCr strain. pcDNA3.0-3D-Flag-HA?eukaryotic?expression vector was constructed via inserting of the full-length 3D gene into pcDNA3.0-Flag-HA. The recombinant plasmid was then transfected into RD cells. After 48h, the RD cells were collected. Western blot test was done to detect 3D proteins. TAP and LC/MS were performed to screen 3D polymerase-interacting proteins. Finally, Co-IP was done to confirm the interaction between CyclinG1 and 3D protein. Results pcDNA3.0-3D-Flag-HA?eukaryotic?expression vector was successfully constructed and 3D proteins were expressed in RD cells. By TAP purification and LC/MS, a number of 3D polymerase-interacting proteins including LATS2, Nek, APC5, Cyclin-G1 and PI3K that involved in apoptosis and regulation of cell cycle were identified. The interaction between CyclinG1 and 3D protein was conf