【摘要】 目的:构建能够在鼻咽癌细胞株中高效表达人Notch1信号胞内段(NIC)的荧光质粒。方法:通过RT-PCR获得人NIC的cDNA;利用基因重组技术插入到pEGFP-N1中;在脂质体介导下转染人低分化鼻咽癌细胞CNE2后,经荧光显微镜、RT-PCR和Western blot检测NIC的表达情况。结果:RT-PCR方法得到一条2424bp的特异性扩增产物,酶切和基因测序结果表明重组质粒构建成功。转染48h后,检测到绿色荧光表达,RT-PCR和Western blot的结果显示NIC的表达量升高。结论:正确构建了人NIC荧光表达质粒。该质粒能够在鼻咽癌细胞株中高效表达,为研究Notch信号通路在鼻咽癌中的作用奠定了实验基础。更多还原

【Abstract】 Objective:To construct expression vectors of notch1 intracellular domain(NIC),using pEGFP-N1 plasmid as mediation.Methods:Extracted the human NIC cDNA cell RNA by using RT-PCR method,then use the gene recombination technology to insert the cDNA into the pEGFP-N1,after transfection human nasopharyngeal carcinoma CNE2 cell by liposome mediated,we used fluorescence microscopy,RT-PCR and Western blot to detect the expression of NIC.Results:By RT-PCR method we got a 2424bp specific amplification products.The enzyme analysis and gene sequencing results showed that the recombinant plasmid build succeed.After transfection for 48h,we detected the expression of green fluorescence,RT-PCR and Western blot results showed the increased expression quantity of NIC.Conclusion:NIC fluorescence expression vector was constructed successfully.The plasmid can efficiently express in nasopharyngeal carcinoma cell lines.It will lay a foundation for the further study of the effective mechanism of Notch signal pathway on nasopharyngeal carcinoma development and progression.更多还原