磁珠法解析奈非那韦抗多发性骨髓瘤作用蛋白  

Study on anti-multiple myeloma related proteins of nelfinavir by magnetic bead separation

作　　者：赵丽 肖小帆 安静林 曾艳 高晓芳 阳泰 ZHAO Li;XIAO Xiaofan;AN Jinglin;ZENG Yan;GAO Xiaofang;YANG Tai(School of Pharmacy,Chengdu Medical College,Chengdu 610500,China)

机构地区：[1]成都医学院药学院,四川成都610500

出　　处：《沈阳药科大学学报》2019年第11期1020-1025,共6页Journal of Shenyang Pharmaceutical University

基　　金：国家自然科学基金资助项目(81201668);成都市科技局项目(2015-HM01-00506-SF,2018-YF05-00454-SN);四川省教育厅重大培育项目(17CZ0011);成都医学院科研基金(CYCG15-01);国家级大学生创新创业训练计划项目(201613705009、201613705070、201613705092).

摘　　要：目的采用磁珠法捕获与奈非那韦抗多发性骨髓瘤(multiple myeloma,MM)相关作用蛋白,并对其进行分析,为探明奈非那韦抗MM作用机制提供理论依据。方法将环氧基磁珠和奈非那韦偶联,然后与MM.1S细胞蛋白裂解产物共孵育,结合奈非那韦作用蛋白,通过缓冲溶液解离奈非那韦偶联蛋白,进行SDS-PAGE(sodium dodecyl sulphate-polyacrylamide gel electrophoresis)电泳和银染分析,对胶上蛋白进行高分辨质谱鉴定,再通过GO(Gene Ontology)数据库和KEGG(Kyoto Encyclopedia of Genes and Genomes)数据库预测蛋白质参与的生物学意义。结果奈非那韦捕获蛋白中可信度较高的蛋白:L-乳酸脱氢酶、核糖体蛋白S3和电压依赖性阴离子选择性通道蛋白1。上述蛋白与肿瘤细胞增殖关系密切。生物信息学分析发现奈非那韦可能和其他潜在生物学功能相关,例如:碳水化合物代谢、脂代谢以及嘌呤代谢等。结论采用一种简单、高效的捕获靶蛋白新方法,为挖掘奈非那韦抗MM作用靶点奠定了理论基础,为药物新靶标筛选和潜在生理功能提供新的思路和方法。Objective To capture the target proteins of nelfinavir on MM.1 S cells by magnetic bead-coupling technology,further to analyze the anti-multiple myeloma related proteins of nelfinavir and provide a theoretical basis to search for the anti-multiple myeloma mechanism of nelfinavir.Methods The epoxy-based magnetic beads w ere coupled w ith nelfinavir,and the magnetic beads-nelfinavir conjugate w as incubated w ith the M M.1 S cell lysate to bind the target proteins of nelfinavir.Then the target proteins were eluted from the magnetic beads-nelfinavir conjugate in buffer solution.Subsequently,the eluted proteins were identified by High resolution mass spectrometry(HRMS)after sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE)system and silver staining analysis.Finally,the target proteins were analyzed by Gene Ontology(GO)database and Kyoto Encyclopedia of Genes and Genomes(KEGG)database.Results Several highly reliable proteins were captured by nelfinavir:L-lactate dehydrogenase,Ribosomal protein S3 and Voltagedependent anion selective channel protein 1,which were closely related to tumor cell proliferation.Furthermore,some other potential biological functions of nelfinavir were revealed via bioinformatics analysis,such as carbohydrate metabolism,lipid metabolism and purine metabolism.Conclusion This study adopts a simpler,more novel and more efficient method to capture target proteins of drugs,which provides a theoretical basis to explore the anti-multiple myeloma related proteins of nelfinavir.It also provides new insights and approaches for the discovery of novel targets and potential functions of drugs.

关 键 词：奈非那韦 磁珠偶联 靶蛋白 银染 生物信息学分析 药物重新定位 

分 类 号：R96[医药卫生—药理学]