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中文摘要：
目的 纯化CaV1.2不同蛋自片段并研究其与CaM的相互作用.方法 质粒转化、筛选得到转化成功BL21菌株,利用IPTG诱导GST钙通道融合蛋白表达,Glutathione-Sepharose 4B beads纯化钙通道不同蛋白片段；采用GST pull-down assay实验研究钙通道蛋白不同片段与CaM以及突变体的相互作用.结果 SDS-PAGE结果显示成功纯化获得钙通道不同蛋白片段；GST pull-down assay结果显示CT1可与CaM及突变体结合,且GST-CT1与野生型CaM结合量显著多于与突变体CaM结合量.结论 成功纯化CaV l.2钙通道蛋白片段并顺利完成GST pull-down assay实验,为深入研究蛋白相互作用或发现新的蛋白相互作用奠定了基础.
英文摘要：
Objective To purify different fragments of CaV1.2 channel and explore its interaction with calmodulin （CaM）.Methods Escherichia coli BL21 competent cells were transformed with pGEX-6p-3 plasmid vector,which were inserted with the cDNAs of CT1,CT2 and CT3.The transformed BL21 cells were incubated,and stimulated with IPTG to express GST fusion proteins.The fusion proteins were purified with Glutathione-Sepharose 4B beads.The interaction of different fragments of CaV1.2 channel with CaM and its mutants was investigated by GST pull-down assay.Results SDS-PAGE showed that proteins of interest were acquired.GST pull-down assay indicated that CT1 could be bound to CaM and its mutants,and the bindings of CaM mutants to CT1 were lower than that of wild CaM.Conclusion Different fragments of CaV1.2 channel were purified successfully and the GST pull-down experiment was completed well,which provided the basis for further researches on protein-protein interactions and development of novel protein interactions.
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