摘要目的 通过对转HBV全基因小鼠肝细胞质膜蛋白质组的分析,研究病毒基因对宿主肝细胞质膜蛋白质表达的影响,探讨HBV感染机制. 方法 以HBV转基因C57小鼠和正常鼠肝为材料,通过蔗糖密度梯度离心结合抗体亲和法纯化肝细胞质膜.用Western blot法验证质膜的纯度和转基因鼠肝细胞质膜中HBV外膜蛋白质的表达.对纯化的质膜组分进行差异蛋白质组学分析. 结果 亲和纯化法较密度梯度离心法能进一步富集质膜近3倍,线粒体的污染减少近2倍;HBV外膜蛋白质只在转基因鼠肝细胞质膜中表达.蛋白质双向电泳分离得到500多个蛋白质点,分析得到26个具有2倍以上差异的蛋白质点,准确鉴定出7个蛋白质.结论 获得了好的质膜纯化方法;HBV基因调节了小鼠肝细胞质膜蛋白质的表达,鉴定得到了与HBV病毒感染相关蛋白质,为进一步探讨HBV感染机制打下了基础.

abstractsObjectives To study liver plasma membrane (PM) proteins affected by hepatitis B virus(HBV), and to study the molecular mechanism of HBV infection through plasma membrane proteome analy-sis of transgene mice livers. Methods Plasma membrane was purified using second antibodysuperparamagnetic beads that combine subcellular fractionation and immunoisolation strategies. Western blotwas used to verify the purification of plasma membrane and the expression of HBV envelope protein. The proteins from plasma membrane were extracted, quantified and separated by two-dimensional electrophoresis.The gels were stained by silver nitrate or Coomassie Blue and analyzed by Imagemaster software. The differ-ent expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF-TOF MS). Results The plasma membrane of mice livers wasenriched 3-fold and the contamination from mitochondria was reduced 2-fold in comparison with the densitygradient centrifugation method. HBV envelope protein was found only in HBV transgene mice livers. Morethan 500 protein spots were separated in Coomassie Blue stained gels. Twenty-six proteins with two-folddifferences were found through lmagemaster software analysis. Seven proteins were identified. ConclusionAn optimized plasma membrane purification method was developed; protein profile of liver plasma mem-brane changed in the transgene mice livers; some proteins related to HBV infection were found, and this workmay be helpful in theresearch of the molecular mechanism of HBV infection.