转乙型肝炎病毒表面抗原基因小鼠肝细胞质膜蛋白质组学研究  

Proteomic analysis of liver plasma membrane from hepatitis B surface antigen transgenic mice

作　　者：贾小芳[1] 李春洪[1] 彭霞[1] 尹林[1] 冯艳玲[2] 马芳[1] 张丽军[1] 

机构地区：[1]上海市公共卫生临床中心科学研究部,201508 [2]上海市公共卫生临床中心病理科,201508

出　　处：《中华传染病杂志》2012年第10期577-582,共6页Chinese Journal of Infectious Diseases

基　　金：王宝恩肝纤维化基金资助项目（20100031）;中央高校基本科研业务费专项资金资助项目（10FX114）

摘　　要：目的分析转HBsAg基因小鼠与正常小鼠肝细胞质膜的差异蛋白质组，为了解乙型肝炎发病机制，寻找药物作用靶标提供指导。方法构建6月龄转HBsAg基因C57小鼠模型，检测转基因组小鼠和正常对照C57小鼠肝脏病理变化。以转基因组和正常对照C57小鼠肝脏为材料，蔗糖密度梯度离心结合磁珠纯化法纯化肝细胞质膜，Western印迹验证纯化的质膜组分纯度，二维凝胶电泳结合ImageMaster软件图像分析质膜蛋白质，获得的差异蛋白质点经胰酶酶切后，进行差异蛋白质的液相色谱串联质谱分析鉴定。结果转HBsAg基因小鼠肝脏呈现肝炎表现，而正常对照C57小鼠肝脏未见异常。蔗糖密度梯度离心结合磁珠纯化法可以有效富集小鼠肝质膜组分，并减少线粒体的污染。小鼠肝脏质膜组分中，共获得30个≥2倍的差异蛋白质点，成功鉴定到11个可能与HBV感染相关的差异蛋白质，其中9个蛋白质在转基因鼠肝质膜中表达上调，2个蛋白质表达下调。这些差异蛋白质包括细胞支架蛋白、心脏钙离子释放通道蛋白、细胞色素B5和ATP合成酶a亚基等。结论本研究鉴定了一批与HBsAg基因表达相关的小鼠肝脏质膜蛋白质，它们可能是乙型肝炎的药物作用靶标，本研究将为进一步探讨HBV感染的机制提供指导。Objective To explore the differential liver plasma membrane （PM） proteins that may be related to the occurrence, development and reversal process of hepatitis and to understand the pathogenesis of hepatitis and the new drug targets by performing a comparative proteomics research of liver PM between hepatitis B surface antigen （HBsAg） transgenic mice and wild-type C57 mice. Methods A 6-month-old HBsAg transgenic mouse model was established. The pathological examination was performed to observe the pathological changes of transgenic mice and wild-type C57 mice. The PM from liver tissue of 6-month-old transgenic mouse and the control mouse were purified through twice sucrose density grade centrifugation combined with second antibody magnetic bead enrichment. The purity of extracted PM was verified by Western blot. Differential proteome expression analysis was performed by using two-dimensional electrophoresis （2-DE） and ImageMaster software analysis. The differentially expressed proteins were lysed by trypsin and identified by liquid chromatography combined with tandem mass spectrometry （LC-MS/MS） analysis. Results The pathological examination results showed that hepatitis was observed in the transgenic mouse group,while no abnormity was found in the controls. The PM was successfully enriched and the mitochondria contamination was reduced by sucrose density grade eentrifugation combined with second antibody magnetic bead purification treatment. Thirty differential mice liver PM protein spots were visualized, in which 11 non-redundant proteins were successfully identified by LC-MS/MS in transgenic mouse group, including 9 up-regulated protein spots and 2 clown-regulated protein spots. These differentially expressed proteins included keratin, cardiac Ca2＋ release channel, cytochrome B5, ATP synthase subunit alpha, etc. Conclusions A batch of HBsAg gene expression related differential proteins are identified in mouse liver plasma. These proteins might be new drug targets for anti-HBV treatment. This s

关 键 词：小鼠 转基因 肝炎表面抗原 乙型 病毒包膜蛋白质类 蛋白质组学 

分 类 号：R51[医药卫生—内科学]