人Notch2受体胞内区基因N2ICD的原核表达、纯化和鉴定  

Prokaryotic expression,purification and identification of intracellular domain gene N2ICD of human Notch2 receptor

作　　者：陈中标[1] 李飞[1] 李佩婷 邱晓媚 叶健斌 胡冰心[1] 宁立军[1] 宁云山[1,2] 李妍[1] 

机构地区：[1]南方医科大学生物技术学院生物治疗研究所,广东广州510515 [2]珠海南医大生物医药公共服务平台有限公司,广东珠海519090

出　　处：《生物技术》2016年第4期335-340,共6页Biotechnology

基　　金：国家高技术研究发展技术(863计划)项目("蛋白质测序新技术新装备及配套试剂国产化";No.2014AA020909);国家自然科学基金-广东省联合基金重点项目("番荔枝内酯防治肿瘤的关键科学问题及纳米靶向制剂成药性研究";No.U1401223);国家自然科学基金面上项目("幽门螺杆菌Lpp20抗原引起的免疫优势CD4+T细胞应答及与胃部疾病的相关性";No.81470831);广东省科技计划创新载体建设项目("珠海南医大生物医药公共服务平台";No.2013B090800036);广东省科技计划项目("医疗器械临床试验服务平台CRO模式";No.2015A040404021;"珠海南医大生物医药公共服务平台有限公司新型研发机构初创期建设补助";No.2016B090919019)

摘　　要：[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 plasmid containing Notch2 intracellular domain gene N21CD and express it in E. coli BL21. [ Methods] Target gene was amplified via PCR from recombinant eukaryotic plasmid pCMV -Tag4/ N21CD, digested by enzymes and cloned into prokaryotic expression vector pGEX - 4T - 1 carrying GST tag. Then the recombi- nant plasmid was transformed into E. coli BL21 and induced by Isopropyl β - D - Thiogalactoside （IPTG）. After the bacterial sediment was lysed by repeating freezing and thawing, lysozyme lysis and ultrasonication, expression form of target protein N21CD was analyzed by SDS - PAGE. The soluble recombinant N21CD was purified by glutathione sepharose 4B. Then Western Bloting was carried out to identify the purified product. [ Results ] Target gene N21CD was obtained via PCR and recombinant plasmid pGEX -4T - 1/N21CD was successfully constructed. Target protein N21CD was expressed in E. coli and fusion protein N21CD/GST was partly expressed in soluble form. Western Bloting suggested that N21CD/GST was highly expressed with rela- tive molecular mass of 110 kDa after being purified. [ Conclusion] Recombinant N21CD was successfully expressed in E. coli, which will lay the foundation for investigating Notch2 receptor＇s role in cell signal transduction.

关 键 词：NOTCH2 N2ICD 克隆 原核表达 纯化 鉴定 

分 类 号：Q291[生物学—细胞生物学]