乙型肝炎病毒基因调控的小鼠肝细胞质膜蛋白质变化  

Proteomic analysis of plasma membrane of HBV transgene mice livers

作　　者：彭霞[1] 贾小芳[1] 周文江[1] 彭建涛[1] 沈燕[1] 刘立文[1] 张丽军[1] 

机构地区：[1]复旦大学附属公共卫生临床中心,上海201508

出　　处：《中华肝脏病杂志》2008年第8期590-593,共4页Chinese Journal of Hepatology

基　　金：国家“863”计划（2006AA02A411）;中国肝炎防治基金会王宝恩肝纤维化研究基金（20070026）;上海市公共卫生临床中心人才引进基金（RCJJP2）.志谢 湖南师大梁宋平教授提供质谱鉴定.

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。Objectives 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 analysis of transgene mice livers. Methods Plasma membrane was purified using second antibody superparamagnetic beads that combine subcellular fractionation and immunoisolation strategies. Western blot was 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 different 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 was enriched 3-fold and the contamination from mitochondria was reduced 2-fold in comparison with the density gradient centrifugation method. HBV envelope protein was found only in HBV transgene mice livers. More than 500 protein spots were separated in Coomassie Blue stained gels. Twenty-six proteins with two-fold differences were found through Imagemaster software analysis. Seven proteins were identified. Conclusion An optimized plasma membrane purification method was developed; protein profile of liver plasma membrane changed in the transgene mice livers; some proteins related to HBV infection were found, and this work may be helpful in theresearch of the molecular mechanism of HBV infection.

关 键 词：肝炎病毒 乙型 蛋白质组 细胞质膜 

分 类 号：R512.6[医药卫生—内科学]