人源性Notch-1胞内段腺病毒载体构建及鉴定

Construction and identification of adenovirus expressing human Notch 1 intracellular domain

目的:构建携带人Notch-1胞内区基因的腺病毒载体(Ad-GFP-NICD),观察其在真核细胞中的表达。方法:通过PCR从pIRES2-EGFP-NICD质粒中扩增目的片段Notch-1胞内区,定向克隆至穿梭质粒载体pShuttle-CMV-EGFP中,经酶切及测序鉴定后,将Notch-1胞内区定向克隆至重组腺病毒骨架载体pAdxsi,构建携带人Notch-1胞内区基因表达盒及绿色荧光蛋白的重组腺病毒载体(pAdxsi-GFP-NICD)。酶切鉴定正确后,转染人胚肾细胞系293细胞,进行重组腺病毒的包装、生产及纯化,半数组织感染量法测定病毒滴度。用重组腺病毒转染人脐静脉内皮细胞,荧光显微镜下观察细胞绿色荧光蛋白的表达,并通过PCR扩增观察细胞Notch-1胞内区的表达。结果:成功构建了携带人Notch-1胞内区的重组腺病毒,纯化后病毒滴度达1.6×1010pfu/mL。腺病毒载体转染人脐静脉内皮细胞后24 h在荧光显微镜下即可观察到绿色荧光蛋白,48 h更为强烈,PCR扩增证实细胞表达Notch-1胞内区。结论:成功构建了携带Notch-1胞内区的重组腺病毒载体,为进一步研究Notch信号通路促进动脉形成的机制研究奠定了实验基础。

Objective To establish the adenovirus vector of human Notch-1 intracellular domain, and to observe its expression in vitro by transfecting human umbilical vein endothelial cell. Methods Plasmid DNA encoding Notch-1 intracellular domain was extracted from the plasmid IRES2-EGFP- Notch-1 intracellular domain by PCR and inserted into the vector of pShuttle-CMV-EGFP. After the confirmation by restriction enzyme digestion and DNA sequencing, the DNA encoding Notch-1 intracellular domain in the new construction was inserted into the vector of recombine plasmid adenovirus and confirmed by restriction enzyme digestion. Then the human embryonic kidney cell line 293 was transfected with correctly identified pAdxsi-GFP-Notch-1 intracellular domain and the recombinant adenovirus were amplified and purified, the virus titer was detected with 50% tissue culture infective dose （TCID50） assay. The expressions of the green fluorescence protein and Notch-1 intracellular domain were detected with fluorescent microscope and PCR in 24 hours after the recombinant adenovirus transfecting human umbilical vein endothelial cell. Results The recombinant adenovirus pAdxsi-GFP-Notch-1 intracellular domain was constructed and amplified with titer of 1.6 × 10^10 pfu/mL. The green fluorescence proteins could be observed with fluorescent microscope in human umbilical vein endothelial cell 24 hours after transfection with a stronger degree after 48 hours, and the expression of Notch-1 intracellular domain was detected by the result of PCR. Conclusion The recombinant adenovirus pAdxsi-GFP-Notch-1 intracellular domain is successfully constructed and this study will lay a foundation for the research on the mechanism of Notch signaling improving artery formation.