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目的构建电压门控性钠通道亚型Na_v1.2IQ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Na_v1.2 IQ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Na_v1.2 IQ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 Na_v1.2IQ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Na_v1.2IQ及其癫痫突变体蛋白,为研究Na_v1.2与相关调节因子在癫痫发病的作用提供理论依据。
Objective To construct plasmids of voltage-gated sodium channel subtype Na_v1.2IQ fragment and its epileptiform mutants, and to prepare high-purity and high-purity in vitro recombinant proteins for their activity identification. Methods The cDNA of Na_v1.2 IQ fragment and epileptic mutants was inserted into pGEX-6p-1 plasmid vector and then transformed into E. coli BL21 competent cells. Na_v1 was induced by isopropylthio-β-D galactoside (IPTG). 2 IQ fragment and epilepsy mutant GST fusion protein expression, GS-4B beads were isolated and purified. SDS-PAGE was used to detect the purity of the target protein and relative molecular mass. The concentration of GST protein was determined by Bradford method. The purified protein was detected by pull-down assay. Results The fragment of Na_v1.2IQ and its epileptic mutants had higher purity and expression in Escherichia coli in vitro and could directly bind with calmodulin in vitro, which proved that the prepared protein fragment had certain activity. Conclusion The stable and highly efficient in vitro recombinant Na_v1.2IQ and its epileptogenic mutant protein were successfully isolated and purified, providing a theoretical basis for the study of the role of Na_v1.2 and related regulators in the pathogenesis of epilepsy.