目的：设计用于表达新型小发卡RNA（shRNA）的慢病毒载体，并考察其用于抗乙型肝炎病毒（HBV）的可行性。方法：对慢病毒载体骨架SapⅠ位点进行突变，并将用于表达新型shRNA的结构亚克隆到改造后的慢病毒载体；设计靶向HBV保守序列的shRNA并克隆到该慢病毒载体，包装含有shRNA序列的重组慢病毒并定量，考察单个及多个shRNA结构串联对慢病毒滴度的影响；将重组慢病毒感染HBV稳定转染肝细胞系HepG2.CW（MOI=3），用杀稻瘟菌素筛选稳定克隆1周后，将其重新消化，并以1×105/孔接种于24孔板，于铺板96 h后取细胞上清检测HBsAg、HBeAg表达水平和HBV DNA的含量。结果：构建了一种用于表达新型shRNA的慢病毒载体，并通过设计靶向HBV保守序列的shRNA实现了HBV复制的高效抑制；发现多个shRNA串联效果优于单个shRNA。结论：构建的新型shRNA慢病毒表达载体可作为防治慢性病毒感染如HBV感染的潜在有效手段之一，值得进一步研究。%Objective: To design and construct a lentiviral vector used for novel small hairpin RNA(shRNA) ex⁃pression, and evaluate its feasibility for anti-hepatitis B virus(HBV) application. Methods: First of all, the SapⅠsites from the lentiviral vector backbone were mutated, and the novel shRNA expression structure was subcloned into the mutated lentiviral vector. Next, shRNA sequences targeting on the conserved HBV sequences were cloned into the lentiviral vector, and the recombinant lentiviral vectors containing shRNA were packaged and quantified to investigate the influence of single or a series of shRNA connected structures on the viral titers. Then the HBV sta⁃bly transfected cell line named HepG2.CW was infected by recombined lentiviruses(MOI=3), and was carefully plated with constant density after 1 week blasticidin selection. Finally, the level of HBsAg, HBeAg and HBV DNA in the cell supernatants were measured respectively after 96 hours. Results: The lentiviral vector used for novel shRNA expression was successfully constructed,and applied for anti-HBV evaluation, whereas the perfor⁃mance of shRNA connected structures is better than the single one. Conclusion: Such novel lentiviral shRNA vec⁃tors can be used as an effective method to prevent chronic viral infections such as HBV, which is worthy for fur⁃ther study.
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