摘要
目的利用免疫共沉淀联合质谱分析的方法,在人胚肺成纤维细胞系MRC-5细胞和胶质瘤细胞系U87MG细胞中,筛选与人巨细胞病毒(HCMV)即刻早期蛋白IE86相互作用的病毒蛋白和剪接相关蛋白。方法通过HCMV感染细胞使IE86蛋白在细胞中表达,采用免疫共沉淀的方法富集两种细胞系中的IE86结合蛋白,SDS-PAGE凝胶电泳分离免疫共沉淀复合物,从胶中切取IE86结合蛋白条带,胶内酶解后进行液相色谱-质谱联用分析及数据库检索,筛选与IE86相互作用蛋白。结果利用免疫共沉淀联合质谱分析,在MRC-5细胞中,共鉴定到与IE86相互作用的病毒编码的蛋白质17种,宿主细胞编码的剪接相关蛋白25种;在U87MG细胞中,共鉴定到病毒编码的蛋白质4种,宿主细胞编码的剪接相关蛋白8种。结论与胶质瘤细胞系U87MG相比,人胚肺成纤维细胞MRC-5中有更多种病毒蛋白及剪接相关蛋白与IE86有相互作用,协同参与IE86的剪接及病毒早、晚期蛋白的表达,这为进一步研究HCMV在不同细胞环境中的复制机制提供了基础。
Objective To identify the viral proteins and splicing-associated proteins interacting with human cytomegalovirus（HCMV）immediate-early protein 86（IE86）using co-immunoprecipitation combined with mass spectrometry in human embryonic lung fibroblasts（MRC-5）and glioma cell line U87 MG. Methods MRC-5 and U87 MG cells were infected with HCMV to induce the expression of IE86 protein.Co-immunoprecipitation was used for the enrichment of IE86 binding proteins in the two cell lines.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to isolate the co-immunoprecipitation complex.Liquid chromatography-mass spectrometry was performed after in-gel digestion for analysis and related databases were searched to identify the proteins interacting with IE86 protein. Results With the help of co-immunoprecipitation and liquid chromatography-mass spectrometry,17 proteins encoded by viruses and 25 splicing-associated proteins encoded by host cells were found to interact with IE86 protein in MRC-5 cells,while 4 proteins encoded by viruses and 8 splicing-associated proteins encoded by host cells were found in U87 MG cells. Conclusion Compared with glioma U87 MG cells,human embryonic lung MRC-5 fibroblasts contain more viral proteins and splicing-associated proteins interacting with HCMV IE86 protein.These proteins may collaboratively participate in IE86 splicing and regulate the expression of early and late proteins in viruses.These results provide a basis for further studies on the replication mechanism of HCMV in different cell environments.
作者
李朝华
胡明
钱冬萌
王斌
LI Zhaohua;HU Ming;QIAN Dongmeng;WANG Bin(Department of Pathogenic Biology, College of Basic Medicine, Qingdao University, Qingdao 266071, China)
出处
《青岛大学学报（医学版）》
CAS
2018年第1期1-5,9,共6页
Journal of Qingdao University(Medical Sciences)
基金
国家自然科学基金面上项目(81471958)
关键词
巨细胞病毒
即早蛋白质类
免疫沉淀法
质谱分析法
蛋白质相互作用
cytomegalovirus： immediate early proteins： immunoprecipitation： mass spectrometry
protein protein interaction
