中文摘要：

目的构建电压门控性钠通道亚型Nav1.2IQ片段及其癫痫突变体的质粒,制备高浓度和高纯度的体外重组蛋白并进行活性鉴定。方法将Nav1.2IQ片段及癫痫突变体的cDNA插入pGEX-6p-1质粒载体后,转化大肠杆菌BL21感受态细胞,利用异丙基硫代-β-D半乳糖苷（IPTG）诱导Nav1.2IQ片段及癫痫突变体GST融合蛋白表达,GS-4B beads进行分离纯化。应用SDS-PAGE检测目的蛋白纯度和相对分子质量;采用Bradford方法测定GST蛋白浓度;应用pull-down法检测纯化后蛋白的活性。结果 Nav1.2IQ片段及其癫痫突变体在体外大肠杆菌中具有较高的纯度和表达量,与钙调蛋白在体外能够直接结合,证明制备的蛋白片段具有一定活性。结论成功分离纯化了稳定高效的体外重组Nav1.2IQ及其癫痫突变体蛋白,为研究Nav1.2与相关调节因子在癫痫发病的作用提供理论依据。

英文摘要：

Objective To establish the prokaryotic expression vector to study the purification and expression and the bioactivity identification of IQ GST fusion proteins and the epileptic mutant relevant to IQ of voltage-gated sodium channel.Methods The Escherichia coli BL21 component cells were transformed with pGEX-6p-l plasmid vector,which was inserted with the cDNAs of IQ and its mutant.The transformed BL21 were incubated,IPTG was applied to stimulate the high expression of GST fusion proteins of IQ and its mutant.The fusion proteins were purified by Glutathione-Sepharose 4B beads,SDS-PAGE analysis was used to detect the purity and relative molecular weight of IQ and its mutant.Bradford method was applied to determine the concentration of purification IQ and its mutant,the protein activity was determined by pull-down method.Results SDS-PAGE showed the high purity of IQ and its mutant which were expressed in Escherichia coli BL21 in vitro,the concentration of IQ and its mutant was 2mg/mL IQ and its mutant could bind with CaM directly by pull-down assay,indicating that the fusion proteins were bioactive.Conclusion IQ and its epileptic mutant fusion proteins were successfully purified and expressed in Escherichia coli BL21,which would provide a basis for further study the effect of Nav1.2 and its relative modulators in epilepsy.