论文部分内容阅读
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.