小鼠水通道蛋白1基因慢病毒载体构建及其在神经膜细胞中的表达

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.

Construction of lentiviral vector containing mouse aquaporin-1 gene and its expression in Schwann cells

Objective To construct a lentiviral vector carrying mouse aquaporin-1 (AQP1) gene and use it for infecting Schwann cells of C57BL/6 mouse, so as to provide AQP1+Schwann cells for further studying the relationship of AQP1 with peripheral nerve system injury edema.Methods Mouse AQP1 gene was inserted into lentiviral vector pCDH-CMV-MCS-EF1-copGFP, and the products were confirmed by PCR and sequencing analysis. pCDH-CMV-MCS-EF1-copGFP-AQP1 and the virus packaging plasmids psPAX2 and pMD were cotransducted into 293T cells to prepare lentivirus-AQP1, and the latter was used to infect C57BL/6 mouse Schwann cells in vitro. The expression of AQP1 mRNA and protein was detected by quantitative real-time PCR and Western blotting analysis in infected mouse Schwann cells. Results PCR and sequencing revealed that the pCDH-CMV-MCS-EF1-copGFP-AQP1 plasmids were successfully constructed. The expression of AQP1 mRNA and protein in Schwann cells was significantly increased than that in cells infected with control lentiviruses (P<0.05). Conclusion We have successfully constructed a recombinant lentivirus carrying AQP1 gene, which can be used to infect mouse Schwann cells, leading to increase of AQP1 mRNA and protein expression.