【摘要】 目的观察细胞液泡蛋白质分选因子4B(VPS4B)显性负性突变体VPS4B-K180Q在体外抑制乙型肝炎病毒(HBV)复制的效应。方法用不同剂量野生型VPS4B真核表达质粒pXF3H-VPS4B和K180Q突变型真核表达质粒pXF3H-K180Q与pHBV1.3共转染Huh7细胞,时间分辨荧光免疫分析法(TRFIA)定量检测细胞培养上清中的HBsAg和HBeAg含量,Southernblot分析细胞内HBV核衣壳相关DNA水平,实时定量PCR检测HBV相关mRNA水平变化。结果野生型VPS4B可抑制HBsAg和HBeAg的分泌,对细胞内HBV核衣壳相关DNA和mRNA的抑制作用较弱;低剂量突变型VPS4可显著抑制HBsAg和HBeAg的分泌,对细胞内HBV核衣壳相关DNA和mRNA的抑制作用较强,且均呈剂量依赖的抑制效应。结论 VPS4B及其显性负性突变体可在体外表现出抗HBV的作用,突变型VPS4B抑制HBV能力高于野生型VPS4B。更多还原

【Abstract】 Objective To investigate the inhibitory effect of vacuolar protein sorting 4B(VPS4B) dominant negative mutant K180Q on hepatitis B virus(HBV) replication in vitro.Methods Huh7 cells were co-transfected with the replication competent 1.3-fold over-length HBV plasmid pHBV1.3,and various amounts of wild-type VPS4B in eukaryotic expression plasmid pXF3H-VPS4B or its dominant negative mutant K180Q in eukaryotic expression plasmid pXF3H-K180Q.HBsAg and HBeAg in the media of the transfected cells were quantitatively determined by time resolved fluorescence immunoassay.DNA related to intracellular HBV core capsid was examined by Southern blot,and mRNA related to HBV was determined by real-time PCR.Results Wild-type VPS4B inhibited the secretion of HBsAg and HBeAg but showed a weak inhibition on the levels of intracellular core-related HBV DNA and mRNA.Comparatively,dominant negative mutant VPS4B-K180Q significantly reduced the secretion of HBsAg and HBeAg at very low concentration,and displayed a does-dependent inhibitory effect on viral DNA and mRNA.Conclusion VPS4B and its dominant negative mutant may possess the effect against HBV in vitro,and the dominant negative mutant appears to be far more potent inhibitory effect than wild-type VPS4B.更多还原