小鼠DLL1真核表达载体的构建及其在肿瘤细胞中的表达  

Construction of pIRES2-EGFP-DLL1 Eukaryotic Vector and Its Expression in Tumor Cells

作　　者：张建平[1] 李娜[2] 王强[1] 亢君君[1] 李永强[1] 迟明[1] 王春梅[1] 

机构地区：[1]第四军医大学基础部中心实验室,陕西西安710032 [2]第四军医大学唐都医院肿瘤科,陕西西安710032

出　　处：《现代生物医学进展》2011年第4期650-652,667,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(30972431)

摘　　要：目的:构建带绿色荧光蛋白的小鼠DLL1全长基因真核表达载体,并在肿瘤细胞中表达。方法:利用PCR特异性引物扩增出DLL1基因全长,将克隆的基因片段插入带绿色荧光蛋白的真核表达载体pIRES2-EGFP质粒中。然后利用脂质体将重组质粒pIRES2-EGFP-DLL1转染进小鼠B16黑色素瘤细胞中,并通过G418筛选后选取生长良好、荧光强度高的三株单克隆进行mRNA水平DLL1表达的鉴定。结果:成功扩增小鼠DLL1的全长基因。克隆入质粒载体后,通过DNA序列测定证实其序列正确。将构建的pIRES2-EGFP-DLL1质粒转染小鼠B16黑色素瘤细胞,经过G418筛选和荧光显微镜观察后,挑选得到GFP阳性率90%以上的稳定转染细胞株。RT-PCR检测稳定转染细胞的mDLL1的表达显著增加,进一步证实了pIRES2-EGFP-DLL1的表达效能。结论:成功构建了小鼠DLL1基因的真核表达质粒,证实其在真核细胞B16中可以表达。Objective：To construct the full-length murine DLL1 eukaryotic expression vector with green fluorescent protein（EGFP） and to express the gene intumor cells.Methods：full-length DLL1 cDNA was synthesized by RT-PCR with specific primers and cloned into pIRES2-EGFP vector to constrcuct recombinant plasmid.The constructed vector was verified and then transfected into murine B16 melanoma cells.The transfected cells were selected with G418 and three green clones were chosen.The expression of DLL1 was detected by RT-PCR at mRNA levels.Results：The full-length DLL1 cDNA was successfully inserted into pIRES2-EGFP eukaryotic vector.B16 melanoma cells were transfected with recombinant pIRES2-EGFP-DLL1 plasmid by liposome.After selected by G418 and fluorescent microscope,clones of which more than 90% cells were green protein positive were obtained.RT-PCR was perform to further analyze mDLL1 expression in transfected cells.Conclusion：pIRES2-EGFP-DLL1 eukaryotic plasmid was successfully constructed and it can express in B16melanoma.

关 键 词：DLL1 肿瘤 质粒构建 

分 类 号：Q75[生物学—分子生物学] Q78