Notch1胞内段荧光表达质粒的构建及鉴定

Construction and identification of fluorescence expression vector of Notch1 intracellular domain

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。

Objective： To construct expression vectors of notchl intracellular domain （ NIC ）, using pEGFP - N1 plasmid as mediation. Methods ：Extracted the human NIC eDNA cell RNA by using RT- PCR method, then use the gene recombination technology to insert the eDNA into the pEGFP - N1, after transfection human nasopharyngeal car- cinoma CNE2 cell by [iposome 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 analy- sis and gene sequencing results showed that the recombinant plasmid build succeed. After transfection for 48h, we de- tected the expression of green fluorescence, RT - PCR and Western blot results showed the increased expression quan- tity 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.