重组AAV1/hFIX病毒制备及其体外转导培养细胞的实验研究

Preparation of rAAV1/hFIX and study of transfected cells in vitro

目的制备携带人凝血因子IX基因的重组AAV1病毒(rAAV1/hFIX),体外转导C2C12细胞并检测hFIX的表达。方法通过"一株载体细胞/一株辅助病毒"的双因素包装策略制备出rAAV1/hFIX,体外转导C2C12细胞后,检测细胞上清中FIX的表达量。结果制备出了高纯度的rAAV1/hFIX病毒,转导C2C12细胞24h后在上清中即可检测到hFIX,连续检测了120h都有表达,24h最高表达量高达到(68.0±3.2)ng/24h。结论制备的rAAV1/hFIX病毒在体外培养细胞中能高水平表达hFIX,为应用基因治疗载体治疗血友病B的体内实验奠定了基础。

[Objective] To prepare the rAAV1/hFIX and assay expression of the hFIX antigen by transfecting C2C12 cell in vitro. [Method] The rAAV-2/hFIX was prepared by ＂one helper virus-one vector cell line＂ strategy and transfeeted C2C12 ceils in vitro and then assayed the hFIX antigen content of cell culture medium. [Result] The hFIX antigen could betested at 24 h after tansfected and also in following 120 h, their expression highest level average reached （68±3.2） ng/24 h. [Conclusion] The rAAV1/hFIX transfected C2C12 cells could efficiently expressed hFIX antigen. The results provided the basis of gene thrapy of hemophilia B by rAAV1.