生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2015年
4期
510-514
,共5页
张婷玉%张秀娟%毛莹莹%胡伟%王学军%王升启
張婷玉%張秀娟%毛瑩瑩%鬍偉%王學軍%王升啟
장정옥%장수연%모형형%호위%왕학군%왕승계
慢病毒载体%小发卡RNA%乙型肝炎病毒
慢病毒載體%小髮卡RNA%乙型肝炎病毒
만병독재체%소발잡RNA%을형간염병독
lentiviral vectors%small hairpin RNA%hepatitis B virus
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。
目的：設計用于錶達新型小髮卡RNA（shRNA）的慢病毒載體，併攷察其用于抗乙型肝炎病毒（HBV）的可行性。方法：對慢病毒載體骨架SapⅠ位點進行突變，併將用于錶達新型shRNA的結構亞剋隆到改造後的慢病毒載體；設計靶嚮HBV保守序列的shRNA併剋隆到該慢病毒載體，包裝含有shRNA序列的重組慢病毒併定量，攷察單箇及多箇shRNA結構串聯對慢病毒滴度的影響；將重組慢病毒感染HBV穩定轉染肝細胞繫HepG2.CW（MOI=3），用殺稻瘟菌素篩選穩定剋隆1週後，將其重新消化，併以1×105/孔接種于24孔闆，于鋪闆96 h後取細胞上清檢測HBsAg、HBeAg錶達水平和HBV DNA的含量。結果：構建瞭一種用于錶達新型shRNA的慢病毒載體，併通過設計靶嚮HBV保守序列的shRNA實現瞭HBV複製的高效抑製；髮現多箇shRNA串聯效果優于單箇shRNA。結論：構建的新型shRNA慢病毒錶達載體可作為防治慢性病毒感染如HBV感染的潛在有效手段之一，值得進一步研究。
목적：설계용우표체신형소발잡RNA（shRNA）적만병독재체，병고찰기용우항을형간염병독（HBV）적가행성。방법：대만병독재체골가SapⅠ위점진행돌변，병장용우표체신형shRNA적결구아극륭도개조후적만병독재체；설계파향HBV보수서렬적shRNA병극륭도해만병독재체，포장함유shRNA서렬적중조만병독병정량，고찰단개급다개shRNA결구천련대만병독적도적영향；장중조만병독감염HBV은정전염간세포계HepG2.CW（MOI=3），용살도온균소사선은정극륭1주후，장기중신소화，병이1×105/공접충우24공판，우포판96 h후취세포상청검측HBsAg、HBeAg표체수평화HBV DNA적함량。결과：구건료일충용우표체신형shRNA적만병독재체，병통과설계파향HBV보수서렬적shRNA실현료HBV복제적고효억제；발현다개shRNA천련효과우우단개shRNA。결론：구건적신형shRNA만병독표체재체가작위방치만성병독감염여HBV감염적잠재유효수단지일，치득진일보연구。
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.
