| 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.小鼠DLL1真核表达载体的构建及其在肿瘤细胞中的表达[J].,2011,11(4):650-652 |
| 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 Expressionin Tumor Cells |
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| DOI： |
| 中文关键词: DLL1 肿瘤 质粒构建 |
| 英文关键词: DLL1 gene Tumor Vector construction |
| 基金项目:国家自然科学基金项目（30972431） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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. |
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