南方医科大学学报

南方医科大学学报 ›› 2015, Vol. 35 ›› Issue (05): 692-.

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Notch1胞内结构域慢病毒表达载体及干扰载体的构建

周学亮，刘季春   

  • 出版日期:2015-05-20 发布日期:2015-05-20

Construction of the N1ICD lentiviral over-expression and interference vectors

  • Online:2015-05-20 Published:2015-05-20

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摘要： 目的构建高滴度大鼠N1ICD慢病毒过表达载体（LV-N1ICD）及N1ICD慢病毒干扰载体（LV-N1ICD-shRNA）。方法以
大鼠cDNA文库为模板，PCR法扩增N1ICD，通过定向克隆构建pGC-FU-N1ICD-3Flag穿梭质粒；设计4对N1ICD-shRNA寡核
苷酸序列，以构建GVC112-N1ICD-shRNA干扰质粒，将pGC-FU-N1ICD-3Flag 和GVC112-N1ICD-shRNA共转293T细胞，检
测Flag 的表达，筛选理想的GVC112-N1ICD-shRNA 干扰质粒。将pGC-FU-N1ICD-3Flag 或GVC112-N1ICD-shRNA 与
pHelper 1.0、pHelper 2.0共转293T细胞，以包装LV-N1ICD和LV-N1ICD-shRNA，分别利用Real-time PCR、药物筛选法进行病
毒滴度测定。LV-N1ICD及LV-N1ICD-shRNA分别感染H9c2 心肌细胞，利用CCK-8 检测细胞活力。结果pGC-FU-N1ICD-
3Flag和GVC112-N1ICD-shRNA质粒经PCR、基因测序及Western-blotting验证构建成功，与pHelper 1.0、pHelper 2.0共转293T
细胞后，取上清浓缩，分别获得高滴度LV-N1ICD 和LV-N1ICD-shRNA。LV-N1ICD 可明显提高心肌细胞活力，
LV-N1ICD-shRNA可降低心肌细胞活力。结论LV-N1ICD和LV-N1ICD-shRNA包装成功，具有Notch1信号通路生物学功能。

Abstract: Objective To construct rat N1ICD lentiviral over-expression vector (LV-N1ICD) and N1ICD lentivirus interference
vector (LV-N1ICD-shRNA. Methods With the rat cDNA as a template, the N1ICD fragment was amplified by PCR to construct
pGC-FU-N1ICD-3Flag shuttle plasmid by directly clone. Four pairs of N1ICD-shRNA oligonucleotide sequences were synthesized
to construct the GVC112-N1ICD-shRNA interference plasmid. pGC-FU-N1ICD-3Flag and GVC112 -N1ICD-shRNA
plasmids were co-transfected into 293T cells to screen for the best interference plasmid in the 4 GVC112-N1ICD-shRNA
plasmids by detecting Flag expression. pGC-FU-N1ICD-3Flag or GVC112-N1ICD-shRNA plasmid along with with pHelper 1.0
and pHelper 2.0 plasmids were co-transfect into 293T cells to package LV-N1ICD and LV-N1ICD-shRNA, and the virus titer
was determined by real-time PCR and drug screening method, respectively. H9c2 cells infected with LV-N1ICD and LVN1ICD-
shRNA respectively were assessed for cell viability using CCK-8 assay. Results pGC-FU-N1ICD-3Flag and GVC112-
N1ICD-shRNA plasmid were verified by PCR, gene sequencing and Western blotting. Co-transfection of the plasmids with
pHelper 1.0, and pHelper 2.0 plasmids into 293T cells obtained high-titer LV-N1ICD and LV-N1ICD-shRNA. LV-N1ICD was
capable of promoting the cell viability and LV-N1ICD-shRNA produced an opposite effect. Conclusion The vectors LV-N1ICD
and LV-N1ICD-shRNA have been successfully constructed and packaged, which have the biological functions of Notch1
signaling.