【作者】 赵艳丰；

【导师】 瞿涤； 周天伦； 叶荣；

【作者基本信息】 复旦大学 ， 病原生物学， 2009， 博士

【摘要】 乙型肝炎病毒(HBV)引起的乙型肝炎是严重危害人类健康的传染病。HBV属于嗜肝DNA病毒科,具有严格宿主特异性,不能感染非灵长类实验动物。目前仍缺乏理想的HBV自然感染细胞培养系统,乙型肝炎病毒和宿主细胞之间的相互作用,病毒感染对宿主细胞的蛋白表达及功能的影响,HBV感染的早期阶段等分子机制目前仍不是十分清楚。鉴于嗜肝DNA病毒具有相似的病毒结构、基因组结构以及复制特点,因此利用鸭乙型肝炎病毒(DHBV)建立的细胞和动物感染模型,用来研究乙型肝炎病毒感染的机制。在体外细胞培养模型中,DHBV能够自然感染鸭原代肝细胞(Primary Duck Hepatocytes, PDHs)并能进行有效的复制,感染的细胞可释放具有感染性的病毒颗粒,因此该感染系统对于研究嗜肝DNA病毒复制过程具有很高的价值。蛋白质组学作为当今生命科学热点与前沿功能基因组学中的重要研究方法,近年来得到了蓬勃的发展,已涉足生命科学中一系列热点领域。蛋白质组学技术的发展推动了病毒学研究的深入,有助于深入了解病毒与宿主相互作用以及引发疾病的机制等。鸡全基因组的测定和蛋白质组学研究技术的发展,为研究DHBV感染对宿主细胞蛋白表达的影响以及寻找与DHBV感染相关的细胞蛋白带来了新的契机。本课题采用鸭原代肝细胞—鸭乙型肝炎病毒(PDHs-DHBV)感染模型,运用蛋白质组学方法分析DHBV感染后鸭肝细胞蛋白表达改变情况,并对病毒感染相关蛋白进行其生物学方面的探讨。本论文在采用PDHs-DHBV感染模型的基础上,利用双向凝胶电泳技术(2-DE),比较分析了DHBV感染后24,72以及120小时的鸭原代肝细胞与未感染的鸭原代肝细胞蛋白差异表达谱,结果显示蛋白表达水平差异1.5倍以上蛋白点有51个。对差异蛋白点进行胶内酶解、肽段提取后,借助基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS/MS)鉴定技术,基于NCBInr数据库,成功鉴定了34个蛋白点,有些胶上的多个蛋白质点对应于同一个蛋白,在合并重复蛋白后得到27个非冗余蛋白。采用生物信息学对差异蛋白的功能进行预测与分类,结果显示差异蛋白主要参与新陈代谢和骨架组成；其余蛋白涉及能量转运、分子伴侣、转录调节、细胞增殖等。为了检验蛋白质组研究结果,分别挑选了病毒感染后鸭肝细胞中丰度上升和下降的蛋白进行验证。其中差异蛋白β-actin及Annexin A2 (ANX2)经Western blot硷证,其变化趋势与双向电泳结果一致。另外(?)amin A,热休克蛋白HSP70以及GAPDH,用人或鼠的多克隆抗体作为一抗,Western blot没有检测到相应条带,可能是由于这些抗体不能与鸭相应蛋白反应所致。本研究发现ANX2在病毒感染鸭原代肝细胞后其表达显著下降。本实验室前期研究,也发现在DHBV易感性降低的鸭原代肝细胞中ANX2表达量显著降低。Annexin A2是膜连蛋白家族的一员,在细胞内可能具有比以往所描述的以钙离子依赖性方式与酸性磷脂结合更为重要而独特的功能。ANX2的生物学功能多种多样,主要包括：(1)参与形成细胞膜的紧密连接；(2)作为细胞表面受体,是组织纤维蛋白溶解酶的细胞受体；(3)参与胞吞和胞吐、形成离子通道。近年来的研究报道显示,ANX2在病毒感染中具有重要的作用。文献报道,在Ca2+存在的条件下,纯化的ANX2可以介导巨细胞病毒(CMV)与磷脂膜的结合,并能够阻断CMV对细胞的感染。在HIV感染中,HIV表面的一种成分能与ANX2结合并促进病毒感染。关于ANX2在嗜肝DNA病毒感染过程中的作用,目前尚未见报道,ANX2在DHBV感染中可能起着一定的作用,需要通过进一步实验证明。为了研究鸭ANX2蛋白在DHBV复制中作用,首先构建了鸭ANX2真核和原核表达质粒,采用鸭ANX2真核表达质粒DNA初步免疫小鼠、原核表达纯化的鸭ANX2蛋白加强免疫的方法,制备了鼠抗鸭ANX2多克隆抗体。利用Western blot验证了对鸭乙型肝炎病毒感染鸭原代肝细胞差异蛋白质组分析发现的差异蛋白ANX2表达的变化,Western blot险测结果显示ANX2蛋白变化趋势与蛋白质组学分析结果一致。为了研究ANX2与DHBVpreS/S的相互作用,将pEGFP-DHBVpreS/S与pDsRed-ANX2重组质粒共转染293T细胞,转染后48小时观察。在激光共聚焦显微镜下观察鸭ANX2可以与DHBVpreS/S共定位于胞浆细胞膜附近。结果提示ANX2与DHBVpreS/S可能存在相互作用,与本实验室前期试验结果符合。进而利用DHBVpreS单克隆抗体以及鼠抗鸭ANX2多克隆抗体,进行免疫共沉淀和pull down实验,证实鸭ANX2与DHBV preS/S蛋白存在相互作用。为明确鸭ANX2对DHBV复制的影响,设计和筛选鸭ANX2 siRNA干扰片段,发现ANX2 siRNA A2-701可以比较明显抑制鸭ANX2的mRNA的转录和蛋白表达。进而用ANX2 siRNA A2-701处理鸭原代肝细胞4天后(确证ANX2的表达下调),再用DHBV感染PDHs,培养4天后,用Dot blot和Southern blot杂交的方法检测DHBV的复制水平。结果显示,siRNA干扰ANX2后,细胞内和细胞培养上清中DHBV DHBV降低。ANX2siRNA干扰初步研究提示ANX2在DHBV的感染过程发挥一定作用。本研究的实验结果丰富了鸭蛋白质数据库,为研究嗜肝DNA病毒的复制、肝炎病毒与宿主细胞相互作用研究奠定了一定的基础。然而,本研究所发现的差异蛋白在嗜肝DNA病毒复制中的作用尚有待于深入研究,随着鸭基因／或基因组和蛋白质数据库的丰富将会推动相关研究。更多还原

【Abstract】 Hepatitis B virus (HBV), belonging to the genus Orthohepadnavirus of the Hepadnaviridae family, is a major cause of liver infection in human. Lack of an appropriate in vitro HBV infection system, how hepatocytes responsing to hepadnaviruses infection remains incompletely understood. Duck hepatitis B virus is a member of the Hepadnaviridae family, classified to Avihepadnavirus, sharing similar biological features with HBV including genome organization and structural organization, viral replication process and biological characteristics. The primary duck hepatocytes-duck hepatitis B virus (PDHs-DHBV) system is a valuable hepadnaviruses infection model in vitro with two major advantages, the ready availability of ducks which are the natural host of DHBV, and the high sensitivity to DHBV of primary hepatocytes from ducklings.With highly reproducible and efficient infection, PDHs-DHBV made the infection system for studying the cellular and molecular biology of hepadnavirus infection under control.The development of proteomic methods revolution has enabled us to assess virus-host interactions and the changes of cellular proteins expression at a global scale, to reveal the connections between virus infection and host cellular functions. How viral infection affects the expression of the cellular proteome has attracted attentions. We intend to utilize a natural PDHs-DHBV infection system to explore hepatocytes response to hepadnaviruses infection. Although Anas platyrhynchos genome has not been sequenced, the decoding of the genome of Gallus gallus (chicken) and the advancement of the genomic techniques bring opportunities to investigate on the interaction of DHBV infection and host protein expression. In the present work, we attempted to analyze primary duck hepatocytes with and without duck hepatitis B virus infection by proteomics and explore the molecular mechanisms of hepadnavirus infections. In the present study, we investigated the effect of DHBV infected primary duck hepatocytes by proteomic analysis. A total of 51 differential expressed protein spots were revealed by 2-DE between DHBV infected and uninfected PDHs at 24,72 and 120h postinfection, respectively. Twenty-seven proteins have been identified by MALDI-TOF MS.Differentially expressed proteins included alpha-enolase, destrin, elongation factor-2, lamin A, heat shock protein, and annexin A2 (ANX2), etc. Western blot further confirmed the down-regulated expression of and ANX2 during DHBV infection. However, duck heat shock protein 70 and lamin A were not detected by Western blot analysis with the rabbit anti-human or anti-mouse heat shock protein 70 polyclonal antibodies, and rabbit anti-human lamin A polyclonal antibody.It was revealed that ANX2 was down-regulated in DHBV infected PDHs comparing with uninfected PDHs. And the proteomics analysis showed that ANX2 expression was down-regulated in PDHs losing their susceptibility to DHBV infection compared to PDHs susceptible to DHBV infection when PDHs cultured in the different stages or culture medium. ANX2, belonging to a family of calcium-dependent, phospholipid binding proteins, is involved in many biological processes such as the Ca2+ dependent exocytosis, calcium transport and cell proliferation. It participates in viral infection, including assisting in the assembly of HIV in monocyte-derived macrophages, as a cellular cofactor supporting HIV-1 infection, enhancing cytomegalovirus binding and membrane fusion. This indicated that ANX2 may be involved in DHBV infection.In order to confirm the ANX2 protein change in the proteomics, we prepared polyclone anti ANX2 antibodies. By Western blot we confirmed the result. To explore the role of ANX2 in the DHBV infection, PDHs were treated with ANX2-specific siRNA (A2-701) or with nontargeting control siRNA (N.C.) and infected with DHBV after the treatment four days. Culture medium was collected and cells were lysed at postinfection (p.i.) four days. Supernatant and intracellular DHBV DNA were analyzed by dot blot hybridization and southern blot hybridization. The results have shown that siRNA of ANX2 in PDHs results in decreased DHBV replication.Recombinant plasmids which express DsRed fusion protein with duck ANX2 and EGFP fusion protein with DHBpreS/S were co-transfected with EGFP-DHBpreS/S fusion protein recombinant plasmid into 293T cell line.The results showed that duck ANX2 co-localized in cytoplasm with DHBVpreS/S by confocal analysis This indicated that ANX2 may interact with DHBVpreS/S. In order to confirm the interaction between them, co-immunoprecipitate and pull down were used. Anti-ANX2 and anti DHBVpreS Western blot showed that DHBVpreS/S coprecipitates with ANX2 from PDHs. Purified ANX2-His protein precipitated on a nickel matrix after incubation with lysates from DHBV infected PDHs and coprecipitated DHBV preS/S. These results supported that ANX2 interacted with DHBV preS/S in PDHs and cell-free systems.In conclusion, this study explored hepatocytes responses to hepadnaviruses infection by cellular proteome analysis, using a natural PDHs-DHBV infection system. The data obtained may lead to better understanding of interactions between hepadnaviruses and hepatocytes and molecular mechanisms of hepadnavirus infection.更多还原