免疫共沉淀联合质谱法筛选和鉴定TS-CBP86-IV相互作用蛋白  

Screening and identification of TS-CBP86-IV interacting proteins using co-immunoprecipitation combining with mass spectrometry

作　　者：申树林[1] 梁季鸿[1] 朱春晖[1] 张迅[1] 刘宁[1] 陆庆华[1] 刘德铭[2] 唐石伏[2] 杨文涛[3] 李群生[3] 

机构地区：[1]广西医科大学第一附属医院男性科,南宁530021 [2]广西柳州市中医院 [3]广西中医药大学附属瑞康医院

出　　处：《中国男科学杂志》2014年第10期7-9,14,共4页Chinese Journal of Andrology

基　　金：国家自然科学基金资助项目(项目编号:81260123);广西自然科学基金资助项目(项目编号:2012GXNSFAA053168)

摘　　要：目的：利用免疫共沉淀联合质谱分析方法，分别从人精子总蛋白和重组BL21细胞总蛋白中筛选和鉴定与TS-CBP86-IV相互作用的蛋白质。方法用克隆技术得到TS-CBP86-IV cDNA编码基因，构建原核表达载体，导入大肠杆菌BL21中诱导表达纯化重组蛋白；另外收集人精子细胞，提取总蛋白，用特异抗体通过免疫共沉淀法分离出TS-CBP86-IV蛋白复合体，再用质谱鉴定可能与其相互作用的蛋白质。结果利用免疫共沉淀联合质谱分析筛选到16个与TS-CBP86相互作用的候选蛋白质，数据库检索发现部分蛋白具有相同功能注释，参与精子运动调控。结论免疫共沉淀联合质谱分析方法成功筛选和鉴定出TS-CBP86-IV相互作用蛋白质，为进一步研究TS-CBP86-IV的功能奠定了良好的基础。Objective To screen and identify interactional proteins with TS-CBP86-IV from total protein of human sperm and Escherichia coli BL21 using co-immunoprecipitation and mass spectrometry. Methods Coding gene of TS-CBP86-IV was cloned and inserted into prokaryotic expression vector, then the recombinant vector was transducted into Escherichia coli for its expression and purification; Human sperm cells were collected and their total protein was extracted. TS-CBP86-IV protein complex was separated by co-immunoprecipitation and the isolated protein was identified by mass spectrometry. Results A total of 16 proteins were identified by co-immunaprecipitaion combining with mass spectrometry. Several proteins identified were associated withregulation of sperm motility. Conclusion Interactional proteins with TS-CBP86-IV were successfully identified by co-immuniprecipitation and mass spectrometry,which establish a solid base for future study of TS-CBP86-IV function.

关 键 词：睾丸特异性钙结合蛋白 免疫沉淀法 质谱分析法 蛋白质相互作用 

分 类 号：R446.121.3[医药卫生—诊断学]