本文選題：登革病毒 + 蚊蟲媒介　； 參考：《南方醫(yī)科大學(xué)》2013年碩士論文

【摘要】：登革病毒是世界100多個國家尤其是熱帶和亞熱帶地區(qū)重要的蟲媒傳染病毒,近乎25-30億人處在潛在感染登革病毒的危險中,每年估計有1億的新增感染者,導(dǎo)致約24000人死亡。目前并無有效疫苗和抗病毒藥物治療。登革病毒屬于黃熱病毒屬中的一個血清型亞群,登革病毒為單股正鏈RNA病毒,其病毒基因組編碼三種結(jié)構(gòu)蛋白(衣殼蛋白C、膜蛋白及其前體M/prM、包膜蛋白E)和七種非結(jié)構(gòu)蛋白(NS1、NS2a、NS2b、NS3、NS4a、NS4b、NS5)。主要由白紋伊蚊和埃及伊蚊傳播,有四種血清型,其中Ⅱ型傳播最廣泛,各型病毒間抗原性有交叉,與乙腦病毒和西尼羅病毒也有部分抗原相同。 病毒受體是由宿主基因組編碼、控制和表達(dá)的一組蛋白質(zhì),它參與病毒與靶細(xì)胞的相互作用,使得病毒能夠吸附于細(xì)胞表面。病毒在受體的介導(dǎo)下最終進(jìn)入細(xì)胞進(jìn)行復(fù)制。細(xì)胞上的病毒受體與病毒的宿主范圍和組織嗜性密切相關(guān)登革病毒是世界范圍的重要的蟲媒傳染病盡管經(jīng)歷了幾十年的研究,然而登革病毒的復(fù)制周期還是未知的。登革病毒可以在多種昆蟲和哺乳動物細(xì)胞培養(yǎng)中增殖,并引起培養(yǎng)細(xì)胞發(fā)生折光性增強(qiáng)、細(xì)胞變圓或細(xì)胞融合等不同程度的細(xì)胞病變。在哺乳類細(xì)胞中的研究報道了大量細(xì)胞表面的登革病毒受體,其與細(xì)胞的種類和病毒的類型有關(guān)。哺乳動物的受體蛋白包括：細(xì)胞連接分子DC-SIGN(凝集素C受體),37/67kDa高親和層粘連蛋白受體,及熱休克蛋白HSP70/90。然而,昆蟲細(xì)胞上的登革病毒受體并沒有鑒定,主要的鑒定僅僅限制在分子量的大小上。然而目前大部分研究都沒有區(qū)別是病毒連接分子還是作用分子,對于篩選出的分子與登革病毒的作用功能研究甚少。 蟲媒媒介效能與產(chǎn)生媒介感染的媒介屏障有關(guān),主要包括中腸感染屏障(midgut infection barrier, MIB)、中腸逃逸屏障(midgut escape barrier, MEB)、唾液腺播散屏障(salivary glands transmission barrier)。如果存在MIB,病毒就不能在蚊蟲中腸細(xì)胞中感染或復(fù)制,這可能是由于細(xì)胞表面缺乏病毒受體。而MEB的存在,使病毒只能在中腸復(fù)制而不能播散,進(jìn)一步感染二級靶器官。迄今為止,蚊傳黃病毒與蚊蟲中腸上皮細(xì)胞受體的相互作用機(jī)制還不十分清楚。本文在研究登革病毒受體的初選時,選用蚊蟲的中腸組織作為主要研究對象,也是基于此媒介屏障的理論。 在選用C6/36細(xì)胞作為潛在登革病毒受體的篩選的載體,研究甚多,研究者利用VOPBA (virus overlay protein binding assay)病毒覆蓋蛋白結(jié)合試驗,將提取的C6/36細(xì)胞膜蛋白通過SDS-PAGE分離,轉(zhuǎn)到固體膜上,再將純化的登革病毒覆蓋結(jié)合蛋白,通過針對登革病毒的單抗識別結(jié)合的病毒間接篩選出特異性蛋白條帶。該方法早期由Mexico等應(yīng)用在白紋伊蚊系C6/36細(xì)胞中篩選登革Ⅱ型病毒潛在受體,篩選出主要的分子為分子量約67000和80000的多肽。這為后來的登革受體研究在方法學(xué)上做了初步的探索,至今研究篩選受體分子應(yīng)用VOPBA作為初步篩選是較為理想的方法,國內(nèi)外研究者外對此方法也不斷改進(jìn),在登革病毒受體研究上取得很大進(jìn)展,用上述方法Munoz等在C6/36細(xì)胞中鑒定出分子量67kDa和80kDa的兩種蛋白為DV2的受體蛋白,尤其是分子量為67kDa的蛋白其多克隆抗體夠抑制DV2連接感染C6/36。之后的研究建議把這兩種蛋白作為四型登革病毒的受體蛋白。Chee等用VOPBA的方法篩選出分子量為48kDa的分子,之后第一次用質(zhì)譜分析的方法鑒定出該分子為微管樣蛋白,微管蛋白在最初并不參與病毒內(nèi)化作用,而是在后階段的內(nèi)化或是在搬運(yùn)病毒顆粒發(fā)揮作用。在早期的研究中Salas-Benito等在C6/36細(xì)胞膜蛋白中鑒定出兩種蛋白40kDa/45kDa,作為DV4的連接分子。并用親和柱色譜法鑒定出這兩種蛋白與重組登革病毒E蛋白相互作用,最終證明這兩種蛋白為糖蛋白,盡管碳水化合物部分并未參與連接作用,之后的研究建議將該蛋白作為熱休克蛋白(Hsp)90。通過抗體介導(dǎo)和小片段RNA介導(dǎo)的干擾下調(diào)抑制素基因的方法來確定抑制素介導(dǎo)病毒進(jìn)入昆蟲細(xì)胞的作用。發(fā)現(xiàn)抑制素的作用只針對登革Ⅱ型病毒,而不參與介導(dǎo)與日本腦炎病毒進(jìn)入細(xì)胞。Paingankar等用VOPBA的方法在C6/36細(xì)胞、埃及伊蚊A7細(xì)胞及白紋伊蚊的中腸緣狀膜組織中鑒定了七種連接蛋白(肌動蛋白、ATP合酶p亞組、熱休克蛋白同系物70、orisis、微管蛋白p鏈、vav-1及抑制素prohibitin)它們共同協(xié)助病毒內(nèi)化和轉(zhuǎn)運(yùn)。在該模型中,作者建議登革病毒和細(xì)胞的初始作用是以非蛋白—蛋白相互作用發(fā)生的包括層粘連蛋白,凝集素,硫酸乙酰肝素或類似的分子,繼而由微管蛋白介導(dǎo)的病毒內(nèi)化。 研究受體介導(dǎo)的登革病毒傳播周期還處在探索階段,至今并未有明確的一套機(jī)制定義該途徑的作用分子,對于受體的研究一直以來都是熱點(diǎn),為今后藥物靶點(diǎn)篩選的研究具有一定參考價值。 目的： 1.應(yīng)用病毒覆蓋結(jié)合蛋白實驗(VOPBA),初步篩選C6/36細(xì)胞膜上與登革病毒作用的連接分子。 2.應(yīng)用病毒覆蓋結(jié)合蛋白實驗(VOPBA),初步篩選白紋伊蚊中腸組織膜蛋白中與登革病毒作用的連接分子。 3.應(yīng)用病毒覆蓋結(jié)合蛋白實驗(VOPBA),比較白紋伊蚊馬氏管組織、卵巢組織、中腸組織與致倦庫蚊中腸組織中登革病毒的連接分子,試圖找出差異性結(jié)果解釋媒介屏障作用。 4.選用二維電泳分離技術(shù)結(jié)合VOPBA,篩選特異結(jié)合分子,應(yīng)用質(zhì)譜分析技術(shù)分析特異蛋白,為該蛋白的功能研究做好鋪墊。 5.設(shè)計埃及伊蚊的陰離子通道蛋白和埃及伊蚊核糖體蛋白S7的real-time PCR引物。 方法： 1.實驗室條件下飼養(yǎng)白紋伊蚊、致倦庫蚊、埃及伊蚊,溫度(26±1)℃,相對濕度70%,光照周期12h：12h(光照：暗室)。用干酵母片飼養(yǎng)蚊幼蟲,定時更換清水,保持水體清潔；成蚊每日飼喂含10%葡萄水,雌蚊在交配后(一般在羽化后三天)可供應(yīng)小白鼠血餐1次,為產(chǎn)卵提供必需營養(yǎng)。 2.C6/36細(xì)胞的培養(yǎng),28℃,于昆蟲細(xì)胞孵箱中培養(yǎng)。 3.昆明乳鼠腦內(nèi)注射接種登革病毒,待乳鼠出現(xiàn)發(fā)病癥狀,收集鼠腦研磨得到含病毒的上清,經(jīng)C6/36細(xì)胞傳代培養(yǎng)獲得滴度更高的病毒液。 4.病毒的滴度測定采用單層細(xì)胞滴定法測定TCID50,即半數(shù)細(xì)胞培養(yǎng)物出現(xiàn)病變的最高稀釋度,采用Reed-Muench法計算TCID50。 5.參考文獻(xiàn)提取C6/36細(xì)胞、白紋伊蚊中腸組織、白紋伊蚊馬氏管、白紋伊蚊卵巢、致倦庫蚊中腸組織的膜蛋白。通過SDS-PAGE分離蛋白,結(jié)和病毒結(jié)合實驗(VOPBA)初篩特異性結(jié)合分子。 6.應(yīng)用二維電泳儀分離C6/36細(xì)胞膜蛋白,結(jié)合病毒結(jié)合試驗(VOPBA)初篩特異性結(jié)合分子,質(zhì)譜分析該特異蛋白的氨基酸序列。 7.利用DNAMAN和PrimerPremier5.0軟件設(shè)計埃及伊蚊陰離子通道蛋白基因埃及伊蚊核糖體蛋白S7的qPCR引物,遵照qPCR引物原則。 8.Trizol法提取埃及伊蚊RNA,利用Takara公司RT-PCR試劑盒擴(kuò)增目的基因。 結(jié)果： 1.登革病毒在白紋伊蚊系細(xì)胞C6/36細(xì)胞中成功復(fù)制,檢測登革病毒滴為107.80TCID50/0.1mL 2.參考文獻(xiàn)提取C6/36細(xì)胞、白紋伊蚊中腸、白紋伊蚊馬氏管、白紋伊蚊卵巢,致倦庫蚊中腸組織膜蛋白經(jīng)SDS-PAGE蛋白膠分離顯示蛋白條帶清晰無降解,可以用于下游實驗。 3.C6/36細(xì)胞膜蛋白病毒孵育組在30kDa有特異性條帶,而陰性對照組無此條帶。 4.研究蚊蟲組織病毒結(jié)合試驗中發(fā)現(xiàn)在白紋伊蚊中腸組織、白紋伊蚊馬氏管帶組織、白紋伊蚊卵巢組織,致倦庫蚊中腸組織都在30kDa有結(jié)合條帶。 5.質(zhì)譜分析結(jié)果比對該蛋白與埃及伊蚊離子通道蛋白的匹配上的氨基酸為29%。 6.應(yīng)用生物信息學(xué)分析,埃及伊蚊陰離子通道蛋白為非跨膜蛋白。 7.成功擴(kuò)增埃及伊蚊離子通道蛋白和埃及伊蚊線粒體蛋白S7基因,長度分別為125bp和247bp,將測序結(jié)果比對原序列確定為目的基因。 結(jié)論： 1.應(yīng)用VOPBA法在C6/36細(xì)胞中篩選出與登革Ⅱ型病毒連接分子,分子量大小為30kDa。 2.應(yīng)用VOPBA法在白紋伊蚊中腸組織、白紋伊蚊馬氏管組織、白紋伊蚊卵巢組織,致倦庫蚊中腸組織都在30kDa都有結(jié)合條帶。 3.質(zhì)譜分析該結(jié)合蛋白與埃及伊蚊離子通道蛋白相似性高,初步鑒定該蛋白為離子通道蛋白,進(jìn)一步對該蛋白進(jìn)行研究。 4.生物信息學(xué)分析該蛋白的性質(zhì)發(fā)現(xiàn)該蛋白為非跨膜蛋白,其參與的作用是連接還是作為伴侶分子有待研究。
[Abstract]:Dengue virus is an important insect borne virus infection virus in more than 100 countries in the world, especially in tropical and subtropical regions. Nearly 25-30 billion people are in the potential to infect the dengue virus. It is estimated that 100 million of the new infected people are infected each year, causing about 24000 deaths. There is no effective vaccine and antiviral treatment. A serotype subgroup in the genus, dengue virus is a single strand of positive chain RNA virus, its viral genome encodes three structural proteins (capsid protein C, membrane protein and its precursor M/prM, envelope protein E) and seven non structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). It is mainly transmitted from Aedes albopictus and Aedes aegypti, with four types of serotypes, of which type II type The most widely spread is the antigenicity of each type of virus, which is the same as that of the Japanese encephalitis virus and Siniro virus.
Virus receptor is a group of proteins that are encoded, controlled and expressed by the host genome. It participates in the interaction between the virus and the target cells, so that the virus can be adsorbed on the cell surface. The virus is finally entered into the cell under the receptor. The virus receptor on the cell is closely related to the host range and the tissue basophilia of the virus. Despite decades of research, the replication cycle of dengue virus is still unknown. Dengue virus can proliferate in a variety of insect and mammalian cells, and cause different degrees of cytopathic changes in cultured cells, such as refractive enhancement, cell circle or cell fusion. Studies in mammalian cells have reported a large number of dengue receptors on the surface of the cells, which are related to the types of cells and the type of the virus. The receptor proteins of mammals include: cell linker DC-SIGN (lectin C receptor), 37/67kDa highly affinity laminin receptor, and heat shock protein HSP70/90., however, on the insect cell The gram virus receptor has not been identified, and the main identification is limited to the size of the molecular weight. However, most of the studies have not been distinguished from the molecular or action molecules of the virus. There is little research on the function of the screened molecules with dengue virus.
The vector efficiency of the insect vector is related to the media barrier that produces vector infection, mainly including the midgut infection barrier (MIB), the midgut escape barrier (midgut escape barrier, MEB), the salivary gland dissemination barrier (salivary glands transmission barrier). This may be due to the lack of virus receptors on the surface of the cells. The presence of MEB makes the virus only replicating in the midgut but not disseminating and further infecting the target organ of the two stage. So far, the interaction mechanism between the mosquito yellows and the midgut epithelial cell receptors of mosquitoes is not very clear. The midgut tissue of mosquitoes is the main research object, and is also based on the theory of the media barrier.
In the selection of C6/36 cells as a potential vector for potential dengue virus receptor, many researchers have used VOPBA (virus overlay protein binding assay) virus covering protein binding test to separate the extracted C6/36 cell membrane protein through SDS-PAGE and turn to solid membrane, then the purified dengue virus covering binding protein, through the needle, through the needle, through the needle, through the needle, through the needle, through the needle The specific protein bands were screened indirectly by the dengue virus (dengue) monoclonal antibody identification combined with the virus. This method was used to screen the potential receptors of dengue type II virus in the C6/36 cells of Aedes albopictus, such as Mexico, and screening the main molecular weight of the polypeptide of about 67000 and 80000 of the molecular weight. This is a methodology for the later dengue receptor study. So far, it is an ideal method to screen the receptor molecule using VOPBA as a preliminary screening. The method has been improved by researchers at home and abroad. Great progress has been made in the study of dengue virus receptor. The two proteins of molecular weight and 80kDa of molecular weight, 67kDa and 80kDa, are identified as DV2 in C6/36 cells. The protein, especially the molecular weight 67kDa, polyclonal antibody that inhibits the DV2 connection infection C6/36., suggests that these two proteins are used as the receptor protein.Chee of the type four dengue virus, and so on. The molecular weight of the molecular weight is screened by VOPBA method, and then the molecular mass analysis method has been used to identify the molecule as microtubule. Like protein, microtubulin was not initially involved in virus internalization, but was internalized in the later stage or played a role in carrying virus particles. In the early study, two proteins, 40kDa/45kDa, were identified in C6/36 cell membrane proteins, 40kDa/45kDa, as a connecting molecule of DV4, and the two proteins were identified by affinity column chromatography. The interaction with the recombinant dengue virus E protein eventually proved that these two proteins were glycoproteins, although the carbohydrate part did not participate in the connection, the subsequent study suggested that the protein was used as a heat shock protein (Hsp) 90. to determine inhibin mediated mediated by antibody mediated and small fragment RNA mediated inhibition of inhibin gene. The effect of the virus into the insect cells was found. It was found that the role of inhibin was only directed against the dengue type II virus, but did not participate in the use of VOPBA to mediate the entry of Japanese encephalitis virus into the cell.Paingankar, and also identified seven connexins (actin, ATP synthase P) in C6/36 cells, Aedes aegypti A7 cells and Aedes albopictus. The group, heat shock protein homologues 70, orisis, tubulin P chain, VAV-1 and inhibin Prohibitin) Co assisted virus internalization and transport. In this model, the author suggested that the initial effect of dengue virus and cells was laminin, lectin, heparin sulfate, or similar fractions occurring in non protein protein interactions. The microtubulin mediated virus internalization.
The study of receptor mediated dengue virus transmission cycle is still in the exploratory stage. There has not been a clear set of mechanisms to define the molecules of this pathway. The research on the receptor has always been a hot spot. It has a certain reference value for the future research of drug target screening.
Objective:
1. using viral coverage binding protein assay (VOPBA), we initially screened the linking molecules of dengue virus on C6/36 cell membrane.
2. using virus coverage binding protein assay (VOPBA), we initially screened the linking molecules between dengue virus and membrane proteins of Aedes albopictus.
3. the use of virus covering binding protein test (VOPBA) was used to compare the martensitic tube tissue of Aedes albopictus, ovarian tissue, midgut tissue and dengue virus in the midgut of Culex pipiens pipiens pipiens pipiens pipiens pipiens, trying to find out the differential results to explain the media barrier.
4. using two-dimensional electrophoresis separation technology and VOPBA, screening specific binding molecules and analyzing specific proteins by mass spectrometry, paving the way for the functional study of the protein.
5. design the real-time PCR primers for the anion channel protein of Aedes aegypti and the Aedes aegypti ribosomal protein S7.
Method錛，

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