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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 eukaryotic expression vector of mouse DLL1 full-length gene with green fluorescent protein and express it in tumor cells. Methods: PCR-specific primers were used to amplify the full length of DLL1 gene. The cloned gene fragment was inserted into the eukaryotic expression vector pIRES2-EGFP plasmid with green fluorescent protein. Then, the recombinant plasmid pIRES2-EGFP-DLL1 was transfected into mouse B16 melanoma cells using liposome, and three monoclonals with good growth and high fluorescence intensity were selected after screening by G418 for mRNA level DLL1 expression. Results: The full-length gene of mouse DLL1 was successfully amplified. After cloning into the plasmid vector, the sequence was confirmed by DNA sequencing. The constructed pIRES2-EGFP-DLL1 plasmid was transfected into mouse B16 melanoma cells. After G418 selection and fluorescence microscopy, stable transfected cell lines with a positive rate of GFP above 90% were selected. The expression of mDLL1 in stably transfected cells was significantly increased by RT-PCR, further confirming the expression efficiency of pIRES2-EGFP-DLL1. Conclusion: The eukaryotic expression plasmid of mouse DLL1 gene was successfully constructed and confirmed to be expressed in eukaryotic cell B16.