人Notch配体DLL4基因的克隆及真核表达  

Cloning and eukaryotic expression of human Notch ligand delta- like ligand 4

作　　者：叶建斌[1] 梁来妹 陈中标[1] 杨宜[1] 宁立军[1] 李妍[1] 宁云山[1,2] 

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》2015年第3期223-226,242,共5页Biotechnology

基　　金：国家高技术研究发展技术(863计划)项目("蛋白质功能分析试剂国产化";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)资助

摘　　要：[目的]构建真核重组质粒DLL4/pCMV-Tag4并进行瞬时表达,为进一步研究DLL4/Notch信号通路奠定基础。[方法]采用PCR的方法扩增人DLL4基因,并克隆于真核表达载体pCMV-Tag4中,通过酶切和测序鉴定后,瞬时转染HEK293 T细胞,荧光定量PCR和Western blot鉴定人DLL4的表达。[结果]成功构建了DLL4/p CMV-Tag4重组质粒并在HEK293T细胞中表达。[结论]人DLL4在HEK293T细胞中表达,为进一步研究DLL4/Notch信号通路奠定基础。[Objective]To construct recombinant eukaryotic expression plasmid DLL4/pCMV-Tag4 and express DLL4 for further studying of DLL4/Notch signaling pathway.[Methods]Human DLL4 gene was amplified by PCR and cloned into eukaryotic expression vector pCMV-Tag4.The recombinant DLL4/pCMV-Tag4 was verified by enzyme restriction and sequencing then transiently and stably transfected it into HEK293 T cells.The real-time quantitative PCR and Western blotting was used to test the expression of human DLL4.[Results]The recombinant eukaryotic expression vector DLL4/pCMV-Tag4 was successfully constructed and DLL4 was expressed in HEK293 T cell.[Conclusion]Human DLL4 was expressed in HEK293 T cell,which provided a foundation for further investigation of DLL4/Notch signaling pathway.

关 键 词：NOTCH信号通路 真核表达 DLL4 pCMV-Tag4 

分 类 号：Q291[生物学—细胞生物学]