宫颈癌HeLa细胞内SKP2结合蛋白的筛选和功能预测  

Screening of S-phase kinase-assuciated protein 2 in the cervical carcinoma Hela cell

作　　者：贾静[1] 方健飞 任娟[1] 贾振宇[1] 王孝举[1] JIA Jing, FANG Jianfei, REN Juan, JIA Zhenyu, WANG Xiaoju(Center for Molecular Medicine, Academy of Medical Scineee of Zhejiang Province, Hangzhou 310013, Zhejiang, Chin)

机构地区：[1]浙江省医学科学院分子医学中心,浙江杭州310013

出　　处：《中国肿瘤生物治疗杂志》2018年第3期258-262,共5页Chinese Journal of Cancer Biotherapy

基　　金：浙江省自然科学基金资助项目(No.LY18H160067;LQ12H16013);浙江省医药卫生科技计划资助项目(No.2015KYB090;2011KYB005)~~

摘　　要：目的:通过免疫共沉淀与质谱分析技术从宫颈癌HeLa细胞中获取与S期激酶相关蛋白2(S-phase kinase-associated protein 2,SKP2)结合的蛋白质群体并预测其生物功能。方法:以免疫共沉淀与Western blotting技术建立SKP2免疫共沉淀体系,以SDS-PAGE和银染技术获得SKP2结合蛋白的特异条带,通过质谱分析技术获得可能与SKP2结合的蛋白群体,应用生物信息学技术对筛选得到的蛋白进行GO分析与KEGG分析。结果:HeLa细胞内存在一定水平的SKP2蛋白表达,可进行免疫共沉淀反应;成功建立了SKP2免疫共沉淀体系,并获得SKP2结合蛋白样品;针对差异的凝胶条带进行质谱分析,共鉴定出SKP2结合蛋白563个;设定筛选条件后,获得可信度较高的SKP2蛋白270个,进行GO分析与KEGG分析后初步预测了结合蛋白参与的细胞功能和信号通路。结论:从宫颈癌HeLa细胞中成功筛选获取SKP2结合蛋白,为后续筛选靶标结合蛋白和寻找细胞靶向药物奠定了基础。Objective：The co-immunoprecipitation and mass spectrometric analysis was carried out to obtain the S-phase kinase-associated protein 2（SKP2）-binding proteins in HeLa cells, and the biological functions of these binding proteins were forecast. Methods：The co-immunoprecipitation system was established by co-immunoprecipitation and Western blotting assay; the specific protein gel of SKP2-binding proteins was obtained by SDS-PAGE and silver staining assay; the potential SKP2-binding proteins was identified by mass spectrometric analysis; and the GO（Gene ontology） analysis and KEGG analysis was carried out by bioinformatics technique.Results： The expression level of SKP2 protein in HeLa cells was high enough for co-immunoprecipitation assay; the co-immunoprecipitation system was established successfully, and SKP2-binding proteins was obtained; a total of 563 proteins were identified by mass spectrometric analysis, and 270 proteins with high credibility were obtained after screening. The GO analysis and KEGG analysis was carried out for the 270 proteins to forecast their functions and pathways. Conclusion： The SKP2-binding proteins were screened successfully, and it was the foundation for the subsequent screening of target-binding proteins and the search for targeting drugs.

关 键 词：细胞S期激酶相关蛋白2 免疫共沉淀 宫颈癌 结合蛋白 

分 类 号：R737.33[医药卫生—肿瘤] R730.5[医药卫生—临床医学]