Pri-miRNA21/23a重组病毒载体的构建及其在肝细胞中的表达鉴定

Construction and Identification of Lentivitus Vectors for Pri-miRNA21/23a in Liver Cells

目的:构建Pri-miRNA-21/23A基因的重组病毒载体,并在L-02肝细胞中获得表达。方法:设计并合成Pri-miRNA-21/23a的基因产物,插入含绿色荧光蛋白Zsgreen基因的真核表达载体pCI Mamma Lian中,以重组质粒为模板,设计扩增含Zsgreen基因的Pri-miRNA序列产物,并将其与pCDH-CMV-MCS-EF1-Puro病毒载体连接,将Pri-miRNA重组病毒载体转染293T细胞后进行病毒包装,病毒上清转染L-02肝细胞后用荧光显微镜及实时荧光定量PCR确认转染效果。结果:荧光定量PCR结果显示重组病毒上清转染L-02肝细胞感染效果良好,经荧光显微镜观察,证实重组病毒载体能在细胞中表达蛋白。结论:构建了Pri-miRNA21/23a重组病毒表达载体并在L-02肝细胞中表达,奠定了miRNA进一步功能研究的基础。

Objective:To construct the lentivitus vectors for Pri-miRNA21/23a and realize the expression in liver cells L-02.Methods:Pri-miRNA-21/23a were designed and synthesized andinserted into pCI MammaLian Expression Vector which contains Zsgreen,from which the Pri-miRNA containing sequence of green fluorescent protein(GFP)was amplified and then linked with pCDH-CMV-MCS-EF1-Puro virus vector.The recombinant lentivirus vector were transfected into293T cells to accomplish virus packaging.Transfection efficiency and protein expression level of virus supernatant transfected L-02liver cell were measured by QPCR and flourescence microscope,respectively.Results:QPCR results confirm the high efficiency transfection of lentivitus vectors.Green flourescence was detected under the inverted flourescence microscope,which shows the stable expression of lentivitus vectors.Conclusion:The successful construction and expression of lentivitus vectors for Pri-miRNA21/23a in L-02liver cells has set foundation for further investigation of miRNA function.