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[目的]构建含Notch2胞内区基因N2ICD的原核重组质粒,并在E.coli中表达。[方法]根据Gen Bank上N2ICD基因序列设计引物,用PCR从真核重组质粒p CMV-Tag4/N2ICD中扩增目的基因,克隆至携带GST标签的pGEX-4T-1原核表达载体中并转化E.coli BL21。异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,收集菌体经反复冻融、溶菌酶破菌、超声破碎后,通过SDS-PAGE电泳检测目的蛋白的表达形式,表达产物经谷胱甘肽琼脂糖树脂glutathione sepharose 4B纯化并经Western Blot鉴定。[结果]PCR获取了目的基因人N2ICD,并成功构建了重组表达质粒pGEX-4T-1/N2ICD,目的基因在E.coli中成功表达并以部分可溶性形式表达在上清中,纯化后经Western Blot鉴定表达产物分子量在110 k Da。[结论]重组N2ICD在E.coli中成功获得可溶性表达,为研究Notch2受体在细胞信号转导中的作用奠定了基础。
[Objective] To construct prokaryotic recombinant plasmids containing Notch2 intracellular domain gene N2ICD and express in E. coli. [Method] According to the sequence of N2ICD gene on GenBank, primers were designed. The target gene was amplified by PCR from pCMV-Tag4 / N2ICD and cloned into pGEX-4T-1 prokaryotic expression vector carrying GST tag and transformed into E.coli .coli BL21. Isopropyl-β-D-thiogalactoside (IPTG) induced expression, the collection of bacteria by repeated freeze-thaw, lysozyme broken, sonicated after SDS-PAGE electrophoresis to detect the expression of the target protein, the expression product Purified with glutathione sepharose 4B and identified by Western Blot. [Result] The recombinant plasmid pGEX-4T-1 / N2ICD was successfully constructed by PCR. The target gene was successfully expressed in E. coli and expressed in partially soluble form in the supernatant. After purification, The molecular weight of Blot was 110 kDa. [Conclusion] Recombinant N2ICD successfully obtained soluble expression in E. coli, which lays the foundation for studying the role of Notch2 receptor in cell signal transduction.