基于逆转录病毒载体的外源基因表达系统的评价和应用

逆转录病毒表达系统是基因治疗研究和RNA干扰技术广泛采用的外源基因表达系统。文中以增强型绿色荧光蛋白 (EGFP) 基因的表达水平和稳定性为指标，比较逆转录病毒表达载体pQCXIN和pcDNA3.1(+) 表达质粒介导的外源基因在HEK293细胞和CHO-K1细胞的表达效率。病毒感染HEK293细胞和CHO-K1细胞的相对荧光强度 (Relative fluorescence intensity，RFI) 均约为对应的质粒转染细胞的2倍。多轮反复感染逆转录病毒表达载体能有效提高HEK293细胞表达EGFP的效率。HEK293细胞经4轮病毒感染后的RFI值较1次病毒感染HEK293细胞的RFI值约提高2倍。此外，逆转录病毒表达载体介导的外源基因表达的稳定性优于质粒转染的外源基因表达。采用携带人重组活性蛋白C (Recombinant human activated protein C，rhAPC) 基因的pQCXIN和HEK293细胞进一步验证了逆转录病毒载体介导的外源基因表达效率，构建了rhAPC表达水平为10~15 mg/(106 cells·d) 的HEK293细胞系。研究结果表明，逆转录病毒表达系统是有应用价值的介导外源基因在哺乳动物细胞高效表达的技术途径。

Evaluation and application of exogenous gene expression system based on retroviral vector

Currently, exogenous gene expression system based on retroviral vector has been widely used as efficient gene expression system in both gene therapeutic research and RNA interference. In this study, we evaluated the efficiency of exogenous gene expression mediated by the retroviral vector in mammalian cells. First, we constructed EGFP (enhanced green fluorescent protein) vector using pcDNA3.1(+) and retroviral vector pQCXIN as backbone vector respectively. Then, we transfected or infected HEK293 cells and CHO-K1 cells with above vector or corresponding retroviral virus, and measured the relative fluorescence intensity (RFI) of EGFP. The results showed that the RFI of the retroviral virus-infected cells was two times higher than that of the plasmid-transfected cells. Further experiments revealed repeated virus infection enhanced the expression of EGFP markedly, with RFI increasing twice after four rounds of virus infection. Furthermore, the EGFP expression in HEK293 cells mediated by the retroviral vector was more stable than transfected with plasmid pcDNA3.1(+). Finally, we further validated the efficiency of exogenous gene expression system based on the retroviral vector by expressing recombinant human activated protein C (rhAPC) in HEK293 cells. We obtained HEK293 cell lines with rhAPC expression between 10 and 15 mg/(106 cells·d). In conclusion, the exogenous gene expression system based on the retroviral vector is an alternative method for the generation of stable and high-expressing mammalian cell lines.