CS_L载体构建表达纯化及活性鉴定  

Construction of CS_L plasmid and expression, purification and activity identification of the recombinant protein

作　　者：雷帅 胡慧媛 王一妃 周诗 苏敬阳 曾晓荣 郝丽英 LEI Shuai;HU Hui-yuan;WANG Yi-fei;ZHOU Shi;SU Jing-yang;ZENG Xiao-rong;HAO Li-ying(Department of PharmaceuticaI Toxicology,School of Pharmacy,China Medical University,Shenyang 110122;The Key Laboratory of Medical Electrophysiology,Ministry of Education of China,Southwest Medical University,Luzhou,646000,China)

机构地区：[1]中国医科大学药学院药物毒理学教研室,辽宁沈阳110122 [2]西南医科大学心血管医学研究所医学电生理学教育部重点实验室,四川泸州646000

出　　处：《解剖科学进展》2018年第6期644-647,共4页Progress of Anatomical Sciences

基　　金：国家自然科学基金(21677030;31471091);国家自然科学基金青年基金(81100108);西南医科大学心血管医学研究所开放基金(201605)

摘　　要：目的构建Calpastatin N末端结构域Calpastatin domain L(CS L)(a.a.150-230)融合蛋白质粒,表达、提取、纯化CS L蛋白并进行生物学活性鉴定。方法将CS L的cDNA片段插入pGEX-6p-3质粒载体后,转化大肠杆菌BL21感受态细胞,大量培养后用异丙基硫代-β-D-半乳糖苷(isopropy-β-D-thiogalactoside, IPTG)诱导蛋白表达,超声破碎法提取CS L蛋白。利用Glutathione-Sepharose 4B (GS-4B)beads和PreScission蛋白酶分离纯化蛋白,pull down assay检测其生物学活性。结果 CS L质粒经测序比对后表明构建成功;超声破碎法提取的CS L蛋白纯度和浓度均较高,并具有能够与GST-CT1融合蛋白浓度依赖性结合的生物学活性。结论成功构建CS L融合蛋白质粒,提取纯化具有生物活性的CS L蛋白,为探讨Cav1.2钙通道自身调节机制奠定基础。Objective To construct the recombinant plasmid vector for N-terminal domain of CSL, and to study the expression and purification and biological activity of CSL recombinant protein. Methods cDNA of CSL was ligated into pGEX-6 p-3 plasmid vector, then transformed into E.coli BL21 competent cells and the transformants were induced with IPTG. The fusion proteins were purified with Glutathione-Sepharose 4 B beads. The biological activity of CSL was analyzed by GST pull-down assay. Results The recombinant plasmid was confirmed with restriction enzyme digestion and sequencing. The concentration and purity of CSL protein were both high enough for the activity detection. And the binding of CSL to CT1 was concentration-dependent. Conclusion The recombinant CSL plasmid was successfully established, which would provide a basis for the future study on the mechanism of auto-regulation of Cav1.2 channel.

关 键 词：钙蛋白酶抑素 pulldownassay 质粒构建 

分 类 号：Q782[生物学—分子生物学]