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为探索慢病毒载体介导的人凝血因子Ⅸ(hFⅨ)cDNA在培养细胞中的表达水平,及重组慢病毒应用于血友病B基因治疗的可行性,分别构建由肝特异性启动子ABP调控的hFⅨ的慢病毒载体FAXW,以及由泛肽-C调控的hFⅨ慢病毒载体FUXW;用磷酸钙法,将慢病毒载体与包装质粒CMV△R8.2和包膜质粒VSV-G共转染产毒细胞293 T,以制备重组慢病毒.重组FUXW病毒分别感染293 T,BHK和L-02细胞,经检测皆有hFⅨ蛋白表达.重组FAXW病毒感染细胞48 h后,仅在L-02细胞检测到hFⅨ高表达(630ng/10~6细胞),而在其他感染细胞中hFⅨ表达较低.将重组FAXW病毒经尾静脉途径注射入血友病B小鼠体内,小鼠血浆中可检测到接近治疗水平的hFⅨ抗原(45 ng/mL),且持续时间超过60 d.上述结果表明,慢病毒载体介导的基因转移为血友病B基因治疗提供了极有潜力的方法.
In order to explore the expression level of lentiviral vector-mediated human coagulation factor Ⅸ (hFⅨ) in cultured cells and the feasibility of recombinant lentivirus for hemophilia B gene therapy, Of lentiviral vector FAXW of hFIX, and hFIX lentiviral vector FUXW regulated by ubiquitin-C; lentiviral vector was cotransfected with packaging plasmid CMVΔR8.2 and envelope plasmid VSV-G by calcium phosphate method 293T cells were infected with recombinant FUXW virus to infect 293T, BHK and L-02 cells respectively.After 48 hours, the cells were infected by recombinant FAXW virus and detected only in L-02 cells (630ng / 10 ~ 6 cells), while hFⅨ was lower in other infected cells.The recombinant FAXW virus was injected into the haemophilia B mice through the caudal vein through the tail vein, and the plasma of mice could be detected close to Therapeutic levels of hFIX antigen (45 ng / mL) persisted for longer than 60 days.The above results indicate that lentiviral vector-mediated gene transfer provides a potentially valuable method for hemophilia B gene therapy.