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

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。

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

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.