SARS冠状病毒与H5N1型禽流感病毒致病机理的研究

【作者】 王洪亮；

【导师】 蒋澄宇；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 近年来,由于哺乳动物细胞表达系统得到越来越广泛的应用,其优点是表达的蛋白质结构正确,且糖基化位点与天然蛋白相似。但哺乳动物细胞表达系统的一个最大瓶颈是产量较低。为了解决这一问题,本论文对几种重要蛋白质的编码序列进行了优化,并将编码序列连接到强表达载体上,转染哺乳动物细胞进行表达,发现所有蛋白质都能够正确表达,证实了序列优化方法的可行性。为了验证序列优化对蛋白表达水平的影响,我们比较了几种蛋白质的表达情况,发现经优化的序列蛋白质表达比未经优化的序列表达能力有明显的提高。而恒定表达细胞株的构建使持续获得高表达量的蛋白质成为可能。为了方便进行蛋白纯化,我们在载体上添加了CD5L引导肽序列,这可以增强蛋白的分泌能力,另外还添加了human Fc标签,可以方便的使用Protein A亲和层析柱进行蛋白富集与纯化。此外,为了能够得到不含标签的蛋白质,目的蛋白与Fc标签之间插入了TEV酶切位点。序列的优化、载体的改进及恒定表达细胞株的构建为获得高产量,高纯度的蛋白质奠定了基础。SARS-CoV进入细胞机制的研究导致严重急性呼吸综合征(SARS)世界性爆发流行的病原体是一种新型的冠状病毒:SARS冠状病毒(SARS-CoV)。SARS是一种以呼吸系统症状为主要表现的全身性疾病,可引起全身多器官多组织损伤,导致患者死亡的主要病因为急性呼吸窘迫综合征(ARDS)。其宿主范围广,传播速度快,且可通过飞沫传播,危害极大。关于这种致命病毒进入宿主细胞的方式,最早有学者通过电镜观察提出病毒通过直接膜融合方式进入细胞。而后来有病毒进入细胞依赖pH的报道,提示病毒进入细胞的方式可能并不是单一的。现有研究证实,SARS-CoV的刺突蛋白(Spikeprotein,S蛋白)是病毒表面最重要的膜蛋白,能够与受体结合并促进病毒入胞。本论文研究发现SARS-CoV可以通过内吞方式进入宿主细胞。SARS-CoV进入宿主细胞的过程中并不依赖于clathrin及caveolin蛋白;而脂筏,这种细胞表面重要的脂质微区在病毒进入细胞的过程中起重要作用。上述结果的阐明为了解SARS-CoV致病机理及研发新型抗病毒药物提供了重要信息。H5N1型禽流感病毒导致急性肺损伤机理的研究最近一段时间以来,不断发生H5N1型禽流感病毒感染禽类和人类的事件。由于这种病毒变异性极高,发生流行的危险较大。而且缺乏有效的预防与治疗方法,病人病死率非常高。因此,H5N1型禽流感病毒致病机理的研究显得尤为重要。病人主要的死亡原因为急性肺损伤,而关于急性肺损伤的分子机理,尚不清楚。本论文研究发现,H5N1型禽流感病毒可以通过以下两种方式导致急性肺损伤:1.H5N1通过诱导肺部巨噬细胞产生ROS,氧化细胞表面磷脂,激活TLR4-TRIF-NFkB通路导致大量炎性因子(IL-6)的产生,导致急性肺损伤。ROS抑制剂及氧化磷脂的清除剂都可以缓解H5N1导致的急性肺损伤。2.H5N1通过Akt-TSC2-mTOR通路诱导细胞产生自噬,并导致细胞死亡,进而导致急性肺损伤的发生。自噬的特异抑制剂及自噬特异分子的敲除可以缓解H5N1导致的急性肺损伤。上述结果的阐明为了解H5N1型禽流感病毒致病机理及研发新型抗病毒药物提供了重要信息。H5N1型禽流感病毒进入宿主细胞机制的研究关于禽流感病毒进入细胞机制的研究主要来自H1N1型病毒,由于不同病毒株进入不同细胞时采取的方式可能有所不同,而H5N1型禽流感病毒近期较为流行,我们研究了H5N1型禽流感病毒进入宿主细胞的方式。本论文发现H5N1型病毒进入人肺上皮细胞A549细胞时,依赖于clathrin蛋白而并不依赖于caveolin蛋白,这一结果的阐明为了解H5N1型禽流感病毒致病机理及研发新型抗病毒药物提供了重要信息。更多还原

【Abstract】 Mammalian expression of codon optimized proteinsThe mammalian expression system has been widely used in recent years due to its ability of producing correctly folded and glycosylated protein.However,one disadvange of this system is the low protein yield.This paper described a new method of codon optimization,which enhances protein expression to a higher level compared with non-optimized sequence.The establishment of a stable expression cell line enables the production of protein in large scale.In order to simplify protein purification,the expression vector was modified by inserting a CD5 lead sequence upstream of coding sequence,which will enhance protein secretion into supernatant,and a human Fc tag,which will facilitate protein purification with a Protein A column.In addition,in order to obtain proteins without tags,a TEV site was constructed between coding sequence and Fc tag.This system enables production of lage-scale,high-purity,and glycosylated proteins.Mechanism of SARS-CoV entry host cellsThe entry pathway of SARS-CoV into host cells has been controversial,direct membrane was firstly observed by electro-micorscope in 2003,however,later,it was reported that the entry is dependent on pH,which suggests that an endocytic pathway may also exist.Our paper found that SARS-CoV can enter via endocytosis and the SARS-CoV was targeted to endosome in Vero E6 cells.We also found that the entry pathway of SARS-CoV into host cells did not dependent on clathrin or caveolae,but, lipid rafts,an important microdomains rich in cholesterol was found to be involved in this process.These findings are of great importance to the development of novel anti-viral drugs.Molecular mechanism of H5N1 avian influenza induced acute lung injuryRecent outbreaks of highly pathogenic avian influenza H5N1 in poultry and humans have raised the concern that a pandemic will occur in the near future.According to the most recent World Health Organization data,reported on March 11,2009,the cumulative mortality attributed to avian influenza HSN1 virus infection is greater than 60%.Acute lung injury(ALI) is the major manifestation associated with patients hospitalized with H5N1 influenza infection.ALI has a high mortality rate and,other than mechanical ventilation and supportive clinical care,there are few specific therapeutic options of proven benefit.We know little about the molecular pathogenesis of ALI.This paper found that H5N1 can cause ALI via two pathways:1.H5N1 induce lung macrophages to produce ROS,which will oxidize phospholipids and generate OxPLs,which will then activate TLR4-TRIF-NFkB pathway,producing large quantity of cytokines,inducing ALI.ROS inhibitors,antibodies that can bind OxPLs can alievate the severity of ALI.2.H5N1 viruses induce autophagy in lung epithelial cells via Akt-TSC2-mTOR pathway.This autophagy leads to cell death,which will cause lung injury.Specific inhibitors and siRNAs targeting autophagy will alievate the severity of ALI.These findings expand our understanding of H5N1 induced lung injury and also provide valuable information for the anti-viral research.Mechanism of H5N1 avian influenza entry host cellsThe mechanism of influenza virus entry has been widely studied;however,all the studies have been carried out with virus strain H1N1,and most of the studies have used the canine kidney cell line MDCK.It has been reported that the influenza virus can enter cells via clathrin-mediated endocytosis,caveolae-mediated endocytosis,or non-clathrin-, non-caveolae-dependent endocytosis,depending on the different cell lines or virus strains used.However,since the risk of an H5N1 pandemic is increasing due to the widespread occurrence of birds infected with H5N1 and the lack of effective drugs or vaccines,it is imperative to understand the mechanism of disease caused by H5N1 in humans.In this paper,we used the H5N1 strain and a cell line of human lung tissue origin,which is the main target of H5N1 infection,and found that the H5N1 virus can enter a human lung carcinoma cell line via clathrin-dependent endocytosis.This finding is relevant for the development of novel therapeutic drugs targeting influenza virus H5N1.更多还原