【作者】 王赛锋；

【导师】 李琦涵； 刘龙丁； 董承红；

【作者基本信息】 中国协和医科大学 ， 免疫学， 2008， 硕士

【摘要】 疱疹病毒对于人类是一种危害严重的病原体,其中以单纯疱疹病毒(Herpessimplex virus,HSV)在人群中的感染最为普遍,HSV原发感染后,机体虽然能够产生特异性免疫反应,但并不能彻底消除病毒,病毒以潜伏感染的形式终生存在于宿主神经节细胞内,在一定状态下可以被重新激活进行裂解性增殖,从而损坏机体上皮组织。单纯疱疹病毒1型(Herpes simplex virus 1,HSV-1)感染能导致一系列的细胞效应,尤其是宿主细胞蛋白质合成途径在病毒侵入后出现明显变化,例如:宿主细胞mRNA被选择性或非特异性降解、mRNA转录被关闭、蛋白质被选择性地降解或保持稳定,甚至重新定义细胞蛋白功能。本课题借助比较蛋白质组学的技术平台,将HSV-1病毒感染的人正常肝细胞L-02与未感染细胞分别进行双向电泳(2-DE),作蛋白质组图,鉴定表达差异较为显著的蛋白点。在所发现的差异蛋白中,有一个为核不均一性核糖核蛋白H2(heterogeneousnuclear ribonucleoprotein H2,hnRNP H2),在细胞中具有正性调控mRNA剪切和加工和转运,当被HSV-1感染后表现为表达量显著下调,这一结果提示:病毒通过影响细胞转录、翻译调控及关闭细胞蛋白质等不同途径实现病毒蛋白的合成。其中对剪接及转运蛋白合成的下调,将直接导致pre-mRNA加工受阻。这对宿主细胞来说,意义显得十分重要。在此基础上,本课题通过Western blot,Northern blot等技术方法进一步验证其在病毒复制过程中不同时段对其表达影响。初步探索hnRNP H2这一重要的mRNA剪切和转运蛋白,在HSV-1感染过程中与病毒蛋白之间的相互作用。NorthernBlot实验则提示,在病毒感染的不同时期hnRNP H2 mRNA的表达量有比较显著的差异,随着病毒感染的进行,表达量显著下降。与此同时,Western blot与Northern Blot实验结果相吻合,即HSV-1感染L-02细胞4h、12h和24h后,检测到hnRNP蛋白随感染进行其表达逐步下调,在0-4h内下降最为明显,从而初步得出结论,可能是由于HSVⅠ的立早基因表达产物的参与,使hnRNP H2在病毒复制早期就开始显著下调。尽管上述细胞蛋白与病毒蛋白的具体作用机制还有待于进一步研究,但本课题为探讨病毒如何改变宿主蛋白合成途径提供了很多新的线索。更多还原

【Abstract】 Herpes virus, as a kind of important pathogen for human, can induce various diseases. Herpes simplex virus (HSV) that is one group of this family induces popular infection in human. In its primary infection, HSV, which is able to induce the specific immune response of individual, can keep its long time existence in neurons of host through latent state and will be reactivated under some certain conditions to induce pathological damages in epidemic tissues.HSV-1 infected cells present a serial changes in micromolecular metabolism, especially in protein synthesis pathway, including degradation of cellular mRNA, shutoff host transcription, selective stabilization and activation of cellular protein, and so on. By used the proteomics platform, normal human liver cell line L-02 and HSV-1 infected L-02 cellular protein mixture were separated individually by 2-DE. A set of protein spots in 2-DE pattern were matched. In this research, it found cellular protein heterogeneous nuclear ribonucleoprotein H2 (hnRNP H2) decreased in HSV-1 infected L-02. hnRNP H2 is a positive regulator in pre-mRNA processing. The changed expression level of this proteins in HSV-1 infected cell may imply that all of them play important roles involving in virus-host interactions to affect the protein expression pattern. in order to verification The effect of expression in different stage of virus infection, we use the Northern blot and Western blot technologies. To further explore the function of the significant splicing protein-hnRNP H2 in the virus infection and interaction with viral protein.By analysis the result, we can draw a conclusion that maybe the participate of the IE expression product, which dramatically effect the expression of hnRNP H2.The changed expression level of this protein in HSV-1 infected cell may imply that all of them play important roles involving in virus-host interactions to affect the protein expression pattern and at the same time will offer us a new clue to analysis the changes of host cell after the infection of virus.更多还原