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

Screening of host proteins interacting with enterovirus 71 3D RNA polymerase by tandem affinity purification

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,48 h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、CyclinG1、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。

Objective To screen host proteins interacting with enterovirus 71（EV71） 3D RNA polymerase by tandem affinity purification（TAP）.Methods The full-length 3D RNA polymerase gene was amplified from the whole genome of EV71 BrCr strain by RT-PCR,and then the fragment was inserted into vector pcDNA3.0-Flag-HA to construct pcDNA3.0-3D-Flag-HA eukaryotic expression vector.The recombinant plasmid was transferred into RD cells,and after 48 h the RD cells were collected.Western blotting was applied to detect the expression of 3D RNA polymerase in the RD cells.TAP and liquid chromatography/mass spectrometry（LC/MS） were performed to screen 3D RNA polymerase-interacting host proteins.Co-immunoprecipitation（Co-IP） was applied to confirm the interaction between candidate proteins（such as cyclin G1） and 3D RNA polymerase.Results Recombinant plasmid pcDNA3.0-3D-Flag-HA was successfully constructed and 3D RNA polymerase was expressed in the RD cells.A number of 3D RNA polymerase-interacting proteins including LATS2,Nek,APC5,Cyclin G1 and PI3K that were involved in cell apoptosis and regulation of cell cycle were identified by TAP purification and LC/MS.The interaction between Cyclin G1 and 3D RNA polymerase was confirmed by Co-IP.Conclusion EV71 3D RNA polymerase may interact with host proteins to induce cell apoptosis and cell cycle changes.