Cav1.2钙通道片段CT1融合蛋白的制备及生物学活性鉴定  

Purification of CT1 fragment of Cav1.2 Channel and Identification of Its Biologic Activity

作　　者：封瑞[1] 胡慧媛[1] 郭凤[1] 于丽凤[1] 赵美眯[1] 龟山正树[2] 郝丽英[1] 

机构地区：[1]中国医科大学药学院药物毒理学教研室,沈阳110001 [2]鹿儿岛大学医学部

出　　处：《中国医科大学学报》2013年第11期970-973,共4页Journal of China Medical University

基　　金：国家自然科学基金(31071004;30870907;81100108)

摘　　要：目的诱导表达Cav1.2钙通道片段CT1与谷胱甘肽转移酶(GST)重组的融合蛋白并进行纯化和鉴定。方法将pGEX6p 3/CT1重组质粒转化入E.coli BL21中并诱导表达,采用非离子去污剂B per的方法分离纯化GST CT1融合蛋白,使用Western blot技术鉴定GST CT1融合蛋白,pull down assay实验方法检测GST CT1融合蛋白的生物学功能。结果非离子去污剂B per能够成功分离纯化GST CT1融合蛋白,识别此片段的特异性抗体能够检测到GST CT1融合蛋白;制备获得的GST CT1融合蛋白具有与钙调蛋白结合的生物学功能。结论技术简单、操作快速的非离子去污剂B per能够分离纯化具有生物学功能的GST CT1融合蛋白。Objective To purify the CT1 fragment of Cavl.2 channel and investigate its biologic activity. Methods Eschedehha coli BI21 was transformed with plasmid of pGEX- 6p-3/CT1. The transformed Escherichia coli BL21 were induced by isopropyl-β-D-thiogalactoside （ling）. Then, the GST-CTI fusion protein was purified by nonionic detergent B-per method, and identified by Western blot. In ＂addition, the pull-down assay was performed to investigate the GST-CT1 fusion protein biologic activity. Results The SDS-PAGE results showed that the GST-CTI fusion protein was successfully purified by nonionic detergent B-per. The Western blot results revealed that the GST-CT1 fusion protein was obtained. Furthermore, the GST-CT1 fusion protein could bind to calmodulin ptotein. Conclusion The GST-CT1 fusion protein was successfully purified by nonionic deter- gent B-per, and the method is simple and rapid.

关 键 词：融合蛋白 非离子去污剂 钙通道 片段 

分 类 号：R965[医药卫生—药理学]