埃博拉病毒入侵宿主細(xì)胞的可視化研究
發(fā)布時間：2018-08-10 07:52
【摘要】：埃博拉病毒(Ebolavirus)是一種烈性出血熱病毒,其在人類中引起的埃博拉出血熱,病死率可高達(dá)90%。[1]埃博拉病毒屬于絲狀病毒科(Flaviviridae)埃博拉病毒屬(Ebolavirus),是一種不分節(jié)段的單股負(fù)鏈RNA病毒,病毒在電子顯微鏡下呈現(xiàn)絲狀結(jié)構(gòu),直徑約80nm,長度在300nm～1500nm之間。埃博拉病毒結(jié)構(gòu)蛋白包括核蛋白NP、包膜蛋白GP、VP30、VP35、基質(zhì)蛋白VP24和VP40以及L(RNA依賴的RNA聚合酶),NP是核衣殼的主要成分,與VP30、VP35和L共同組成核蛋白復(fù)合物(RNP complex),負(fù)責(zé)病毒的復(fù)制與轉(zhuǎn)錄。病毒樣顆粒(Virus-like particals)是不含病毒基因組的空殼或包膜狀顆粒結(jié)構(gòu)。本實(shí)驗通過構(gòu)建和表達(dá)埃博拉病毒樣顆粒(Ebola Virus like particals,VLP)和復(fù)制型埃博拉病毒樣顆粒(Replication and transcription-competent virus like particles,trVLP),并分別對埃博拉病毒樣顆粒(VLP)和復(fù)制型埃博拉病毒樣顆粒(trVLP)進(jìn)行熒光標(biāo)記(VLP使用插入熒光蛋白的方法,trVLP使用蛋白質(zhì)熒光標(biāo)記試劑盒),使其帶有熒光,為下一步實(shí)時動態(tài)研究埃博拉病毒入侵細(xì)胞的機(jī)制提供了基礎(chǔ)。[2]本研究的主要內(nèi)容包括:1.埃博拉病毒VLP/trVLP的表達(dá)和鑒定本部分研究工作通過表達(dá)埃博拉病毒的結(jié)構(gòu)蛋白并瞬時轉(zhuǎn)染細(xì)胞來組裝不含有病毒核酸的病毒樣顆粒,其中埃博拉病毒樣顆粒(VLP)是利用埃博拉病毒的基質(zhì)蛋白VP40和包膜糖蛋白GP共同轉(zhuǎn)染293T細(xì)胞所得,復(fù)制型埃博拉病毒樣顆粒(trVLP)是以基質(zhì)蛋白VP40、VP24以及糖蛋白GP作為包裝主體,在其他蛋白如VP35、VP30、NP、L等的幫助下表達(dá)組裝具有轉(zhuǎn)錄和復(fù)制能力的病毒樣顆粒,稱為復(fù)制型埃博拉病毒樣顆粒(trVLP)。病毒樣顆粒表達(dá)后,利用透射電子顯微鏡觀察確定VLP/trVLP類似于天然埃博拉病毒的絲狀結(jié)構(gòu),從形態(tài)學(xué)上證明了病毒樣顆粒表達(dá)的成功。對于trVLP,由于其基因組中含有一個報告基因,利用海腎熒光素酶檢測系統(tǒng)對報告基因的表達(dá)進(jìn)行檢測,陽性檢測值證明trVLP的成功表達(dá)。此外,埃博拉VLP中由于插入了綠色熒光蛋白,表達(dá)成功后在488nm激光下能夠發(fā)出綠色熒光,在熒光顯微鏡下可以觀察到明顯的綠色熒光信號。而對于trVLP使用蛋白質(zhì)標(biāo)記試劑盒對其進(jìn)行熒光標(biāo)記,使其帶有綠色熒光標(biāo)記。將VLP或者標(biāo)記后的trVLP加入到Vero細(xì)胞中吸附一段時間,在熒光顯微鏡下可見病毒樣顆�？梢晕揭约斑M(jìn)入細(xì)胞,證明了表達(dá)的VLP和trVLP具有進(jìn)入細(xì)胞的能力,為進(jìn)一步研究埃博拉病毒入侵細(xì)胞的機(jī)制提供了基礎(chǔ)。2.研究埃博拉病毒與細(xì)胞膜脂筏的相互作用在本實(shí)驗中,對埃博拉病毒樣顆粒(VLP)和復(fù)制型埃博拉病毒樣顆粒(trVLP)分別采用重組插入熒光蛋白技術(shù)和蛋白質(zhì)標(biāo)記方法進(jìn)行熒光標(biāo)記,對細(xì)胞膜脂筏進(jìn)行脂筏特異性熒光染料的標(biāo)記,運(yùn)用PEUltraVIEW VoX雙碟片活細(xì)胞熒光共聚焦顯微鏡設(shè)置包括明場在內(nèi)的多個通道,在同一視野下觀察不同通道激發(fā)的熒光信號。通過應(yīng)用這些技術(shù),可以實(shí)現(xiàn)埃博拉病毒入侵細(xì)胞過程的可視化研究。本研究發(fā)現(xiàn)埃博拉病毒樣顆粒(VLP)和復(fù)制型埃博拉病毒樣顆粒(trvLP)都可以吸附并進(jìn)入細(xì)胞內(nèi),而且兩者都與細(xì)胞膜的脂筏結(jié)構(gòu)存在共定位現(xiàn)象。通過實(shí)時動態(tài)研究還發(fā)現(xiàn)細(xì)胞膜脂筏參與了埃博拉病毒進(jìn)入細(xì)胞的過程,證明埃博拉病毒進(jìn)入細(xì)胞的過程中存在與脂筏的相互作用。進(jìn)一步對細(xì)胞進(jìn)行脂筏抑制劑(甲基-β-環(huán)糊精)處理,發(fā)現(xiàn)經(jīng)過相同吸附時間,病毒進(jìn)入細(xì)胞的效率呈現(xiàn)明顯下降,從而證明脂筏在埃博拉病毒入侵細(xì)胞的過程中發(fā)揮了重要作用。提示埃博拉病毒可能通過脂筏途徑進(jìn)入細(xì)胞,為研究鑒定新型藥物靶點(diǎn)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.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R373
本文編號：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.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R373
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