埃博拉病毒入侵宿主细胞的可视化研究
发布时间：2018-08-10 07:52
【摘要】：埃博拉病毒(Ebolavirus)是一种烈性出血热病毒,其在人类中引起的埃博拉出血热,病死率可高达90%。[1]埃博拉病毒属于丝状病毒科(Flaviviridae)埃博拉病毒属(Ebolavirus),是一种不分节段的单股负链RNA病毒,病毒在电子显微镜下呈现丝状结构,直径约80nm,长度在300nm～1500nm之间。埃博拉病毒结构蛋白包括核蛋白NP、包膜蛋白GP、VP30、VP35、基质蛋白VP24和VP40以及L(RNA依赖的RNA聚合酶),NP是核衣壳的主要成分,与VP30、VP35和L共同组成核蛋白复合物(RNP complex),负责病毒的复制与转录。病毒样颗粒(Virus-like particals)是不含病毒基因组的空壳或包膜状颗粒结构。本实验通过构建和表达埃博拉病毒样颗粒(Ebola Virus like particals,VLP)和复制型埃博拉病毒样颗粒(Replication and transcription-competent virus like particles,trVLP),并分别对埃博拉病毒样颗粒(VLP)和复制型埃博拉病毒样颗粒(trVLP)进行荧光标记(VLP使用插入荧光蛋白的方法,trVLP使用蛋白质荧光标记试剂盒),使其带有荧光,为下一步实时动态研究埃博拉病毒入侵细胞的机制提供了基础。[2]本研究的主要内容包括:1.埃博拉病毒VLP/trVLP的表达和鉴定本部分研究工作通过表达埃博拉病毒的结构蛋白并瞬时转染细胞来组装不含有病毒核酸的病毒样颗粒,其中埃博拉病毒样颗粒(VLP)是利用埃博拉病毒的基质蛋白VP40和包膜糖蛋白GP共同转染293T细胞所得,复制型埃博拉病毒样颗粒(trVLP)是以基质蛋白VP40、VP24以及糖蛋白GP作为包装主体,在其他蛋白如VP35、VP30、NP、L等的帮助下表达组装具有转录和复制能力的病毒样颗粒,称为复制型埃博拉病毒样颗粒(trVLP)。病毒样颗粒表达后,利用透射电子显微镜观察确定VLP/trVLP类似于天然埃博拉病毒的丝状结构,从形态学上证明了病毒样颗粒表达的成功。对于trVLP,由于其基因组中含有一个报告基因,利用海肾荧光素酶检测系统对报告基因的表达进行检测,阳性检测值证明trVLP的成功表达。此外,埃博拉VLP中由于插入了绿色荧光蛋白,表达成功后在488nm激光下能够发出绿色荧光,在荧光显微镜下可以观察到明显的绿色荧光信号。而对于trVLP使用蛋白质标记试剂盒对其进行荧光标记,使其带有绿色荧光标记。将VLP或者标记后的trVLP加入到Vero细胞中吸附一段时间,在荧光显微镜下可见病毒样颗粒可以吸附以及进入细胞,证明了表达的VLP和trVLP具有进入细胞的能力,为进一步研究埃博拉病毒入侵细胞的机制提供了基础。2.研究埃博拉病毒与细胞膜脂筏的相互作用在本实验中,对埃博拉病毒样颗粒(VLP)和复制型埃博拉病毒样颗粒(trVLP)分别采用重组插入荧光蛋白技术和蛋白质标记方法进行荧光标记,对细胞膜脂筏进行脂筏特异性荧光染料的标记,运用PEUltraVIEW VoX双碟片活细胞荧光共聚焦显微镜设置包括明场在内的多个通道,在同一视野下观察不同通道激发的荧光信号。通过应用这些技术,可以实现埃博拉病毒入侵细胞过程的可视化研究。本研究发现埃博拉病毒样颗粒(VLP)和复制型埃博拉病毒样颗粒(trvLP)都可以吸附并进入细胞内,而且两者都与细胞膜的脂筏结构存在共定位现象。通过实时动态研究还发现细胞膜脂筏参与了埃博拉病毒进入细胞的过程,证明埃博拉病毒进入细胞的过程中存在与脂筏的相互作用。进一步对细胞进行脂筏抑制剂(甲基-β-环糊精)处理,发现经过相同吸附时间,病毒进入细胞的效率呈现明显下降,从而证明脂筏在埃博拉病毒入侵细胞的过程中发挥了重要作用。提示埃博拉病毒可能通过脂筏途径进入细胞,为研究鉴定新型药物靶点提供了新的思路。
[Abstract]:Ebora virus (Ebolavirus) is a strong hemorrhagic fever virus, the Ebora hemorrhagic fever caused in human, the fatality rate can be as high as 90%.[1] Ebora virus belonging to the filiform virus family (Flaviviridae) Ebora virus (Ebolavirus), is an insegmental single strand of negative chain RNA virus, the virus is filamentous under the electron microscope. The diameter is about 80nm, and the length is between 300nm and 1500nm. The Ebola virus structural proteins include nuclear protein NP, membrane protein GP, VP30, VP35, matrix protein VP24 and VP40, and L (RNA dependent RNA polymerase). Virus-like particals is an empty shell or capsule like granular structure without the virus genome. This experiment was constructed and expressed by Ebola like particles (Ebola Virus like particals, VLP) and replicative Ebola virus like particles (Replication and transcription-competent virus like), and respectively to Ebola. VLP and replicative Ebola virus like particles (trVLP) are marked by fluorescence labeling (VLP is used to insert fluorescent protein, trVLP uses a protein fluorescent labeling kit) to make it fluorescent. The main contents of the basic.[2] study for the next step of real-time dynamic study of Ebola virus invading cells are the main contents of this study. 1. Ebola virus VLP/trVLP expression and identification in this part of the work by expressing Ebola virus structure protein and transient transfection of cells to assemble virus like particles that do not contain viral nucleic acid, in which Ebola virus like particles (VLP) are co transfected to 293T cells using Ebola virus matrix protein VP40 and envelope glycoprotein GP It is obtained that the replicative Ebola virus like particles (trVLP) are packaged with matrix protein VP40, VP24 and glycoprotein GP as the main body of the virus like particles, called the replicative Ebola virus like particles (trVLP), with the help of other proteins such as VP35, VP30, NP, L, etc., which are called the replicative Ebola virus like particles (trVLP). An electron microscope is used to determine the filamentous structure of VLP/trVLP similar to the natural Ebola virus. It has proved the success of the expression of virus like particles in morphology. For trVLP, the expression of the reporter gene is detected by the luciferase detection system of the sea kidney because of its genome, and the positive detection value proves that trVLP In addition, after the insertion of green fluorescent protein in Ebola VLP, a green fluorescence can be produced under the 488nm laser after the expression is successful, and a clear green fluorescence signal can be observed under the fluorescence microscope. And trVLP is marked with a green fluorescent marker with a protein marker kit. VLP or The trVLP after labeling was added to Vero cells for a period of time. Under the fluorescence microscope, the virus like particles could be adsorbed and entered into cells. It was proved that the expression of VLP and trVLP had the ability to enter the cells. The basic.2. study of Ebola virus and cell membrane lipid for the further study of the mechanism of Ebola virus invading cells The interaction of rafts in this experiment is to label Ebola virus like particles (VLP) and replicative Ebola virus like particles (trVLP), using recombinant insertion fluorescent protein technique and protein labeling method respectively, labeling the lipid rafts of cell membrane rafts with lipid rafts specific fluorescent dyes, and using PEUltraVIEW VoX double disc live cell fluorescing. The optical confocal microscope sets a number of channels, including the bright field, to observe the fluorescence signals excited by different channels in the same field. By using these techniques, we can visualize the process of Ebola virus invading cells. This study found that Ebola virus like particles (VLP) and replicative Ebola virus like particles (trvLP) It can be adsorbed and entered into the cell, and both have co localization with the lipid rafts of the cell membrane. The preparation (methyl - beta cyclodextrin) treatment showed that the efficiency of virus entering cells decreased significantly after the same adsorption time, which showed that the lipid rafts played an important role in the process of Ebola virus invasion, suggesting that Ebola virus may enter cells through lipid rafts, providing new targets for the identification of new drug targets. Thinking.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373
本文编号：2175436
[Abstract]:Ebora virus (Ebolavirus) is a strong hemorrhagic fever virus, the Ebora hemorrhagic fever caused in human, the fatality rate can be as high as 90%.[1] Ebora virus belonging to the filiform virus family (Flaviviridae) Ebora virus (Ebolavirus), is an insegmental single strand of negative chain RNA virus, the virus is filamentous under the electron microscope. The diameter is about 80nm, and the length is between 300nm and 1500nm. The Ebola virus structural proteins include nuclear protein NP, membrane protein GP, VP30, VP35, matrix protein VP24 and VP40, and L (RNA dependent RNA polymerase). Virus-like particals is an empty shell or capsule like granular structure without the virus genome. This experiment was constructed and expressed by Ebola like particles (Ebola Virus like particals, VLP) and replicative Ebola virus like particles (Replication and transcription-competent virus like), and respectively to Ebola. VLP and replicative Ebola virus like particles (trVLP) are marked by fluorescence labeling (VLP is used to insert fluorescent protein, trVLP uses a protein fluorescent labeling kit) to make it fluorescent. The main contents of the basic.[2] study for the next step of real-time dynamic study of Ebola virus invading cells are the main contents of this study. 1. Ebola virus VLP/trVLP expression and identification in this part of the work by expressing Ebola virus structure protein and transient transfection of cells to assemble virus like particles that do not contain viral nucleic acid, in which Ebola virus like particles (VLP) are co transfected to 293T cells using Ebola virus matrix protein VP40 and envelope glycoprotein GP It is obtained that the replicative Ebola virus like particles (trVLP) are packaged with matrix protein VP40, VP24 and glycoprotein GP as the main body of the virus like particles, called the replicative Ebola virus like particles (trVLP), with the help of other proteins such as VP35, VP30, NP, L, etc., which are called the replicative Ebola virus like particles (trVLP). An electron microscope is used to determine the filamentous structure of VLP/trVLP similar to the natural Ebola virus. It has proved the success of the expression of virus like particles in morphology. For trVLP, the expression of the reporter gene is detected by the luciferase detection system of the sea kidney because of its genome, and the positive detection value proves that trVLP In addition, after the insertion of green fluorescent protein in Ebola VLP, a green fluorescence can be produced under the 488nm laser after the expression is successful, and a clear green fluorescence signal can be observed under the fluorescence microscope. And trVLP is marked with a green fluorescent marker with a protein marker kit. VLP or The trVLP after labeling was added to Vero cells for a period of time. Under the fluorescence microscope, the virus like particles could be adsorbed and entered into cells. It was proved that the expression of VLP and trVLP had the ability to enter the cells. The basic.2. study of Ebola virus and cell membrane lipid for the further study of the mechanism of Ebola virus invading cells The interaction of rafts in this experiment is to label Ebola virus like particles (VLP) and replicative Ebola virus like particles (trVLP), using recombinant insertion fluorescent protein technique and protein labeling method respectively, labeling the lipid rafts of cell membrane rafts with lipid rafts specific fluorescent dyes, and using PEUltraVIEW VoX double disc live cell fluorescing. The optical confocal microscope sets a number of channels, including the bright field, to observe the fluorescence signals excited by different channels in the same field. By using these techniques, we can visualize the process of Ebola virus invading cells. This study found that Ebola virus like particles (VLP) and replicative Ebola virus like particles (trvLP) It can be adsorbed and entered into the cell, and both have co localization with the lipid rafts of the cell membrane. The preparation (methyl - beta cyclodextrin) treatment showed that the efficiency of virus entering cells decreased significantly after the same adsorption time, which showed that the lipid rafts played an important role in the process of Ebola virus invasion, suggesting that Ebola virus may enter cells through lipid rafts, providing new targets for the identification of new drug targets. Thinking.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373
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