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目的构建表达小鼠水通道蛋白1(AQP1)基因的慢病毒载体,体外感染原代培养的C57BL/6小鼠神经膜细胞,观察是否提高AQP1的表达,为进一步研究AQP1与周围神经系统损伤后水肿的关系奠定基础。方法将小鼠AQP1基因克隆到慢病毒pCDH-CMV-MCS-EF1-copGFP载体,通过PCR和测序鉴定获得连接正确的克隆。将鉴定后的重组表达质粒pCDH-CMV-MCS-EF1-copGFP-AQP1与包装质粒psPAX2、pMD共转染293T细胞,制备携带AQP1基因的慢病毒lentivirus-AQP1。体外培养C57BL/6小鼠的神经膜细胞,将lentivirus-AQP1感染神经膜细胞,RT-PCR和蛋白质印迹法检测感染后神经膜细胞AQP1 mRNA和蛋白的表达情况。结果构建的慢病毒载体pCDH-CMV-MCS-EF1-copGFP-AQP1经PCR鉴定和测序正确。慢病毒lentivirus-AQP1感染神经膜细胞后AQP1表达增加(P<0.05)。结论成功构建了小鼠AQP1基因的慢病毒表达载体,该载体能有效感染神经膜细胞,使AQP1 mRNA和蛋白表达水平增高。
Objective To construct a lentiviral vector expressing mouse aquaporin 1 (AQP1) gene and infect primary cultured C57BL / 6 mouse mesenchymal cells in vitro to observe whether it enhances the expression of AQP1. To further study the effect of AQP1 on the peripheral nervous system The relationship between edema laid the foundation. Methods Mouse AQP1 gene was cloned into the lentiviral vector pCDH-CMV-MCS-EF1-copGFP. The correct clones were obtained by PCR and sequencing. The identified recombinant plasmid pCDH-CMV-MCS-EF1-copGFP-AQP1 and the packaging plasmid psPAX2, pMD were co-transfected 293T cells lentivirus-AQP1 carrying AQP1 gene. Cultured in vitro C57BL / 6 mouse neurofilm cells, the lentivirus-AQP1 infection of the membrane cells, the expression of AQP1 mRNA and protein expression of the membrane after infection by RT-PCR and Western blot. Results The lentiviral vector pCDH-CMV-MCS-EF1-copGFP-AQP1 was identified by PCR and sequenced correctly. The lentivirus-AQP1 lentivirus-infected AQP1 expression was increased (P <0.05). Conclusion The lentiviral expression vector of mouse AQP1 gene was successfully constructed. The vector can effectively infect the mesothelial cells and increase the expression of AQP1 mRNA and protein.