蚊虫细胞与蚊虫组织中登革病毒受体分子筛选与鉴定

发布时间：2018-05-04 21:45

  本文选题：登革病毒 + 蚊虫媒介　； 参考：《南方医科大学》2013年硕士论文

【摘要】：登革病毒是世界100多个国家尤其是热带和亚热带地区重要的虫媒传染病毒,近乎25-30亿人处在潜在感染登革病毒的危险中,每年估计有1亿的新增感染者,导致约24000人死亡。目前并无有效疫苗和抗病毒药物治疗。登革病毒属于黄热病毒属中的一个血清型亚群,登革病毒为单股正链RNA病毒,其病毒基因组编码三种结构蛋白(衣壳蛋白C、膜蛋白及其前体M/prM、包膜蛋白E)和七种非结构蛋白(NS1、NS2a、NS2b、NS3、NS4a、NS4b、NS5)。主要由白纹伊蚊和埃及伊蚊传播,有四种血清型,其中Ⅱ型传播最广泛,各型病毒间抗原性有交叉,与乙脑病毒和西尼罗病毒也有部分抗原相同。 病毒受体是由宿主基因组编码、控制和表达的一组蛋白质,它参与病毒与靶细胞的相互作用,使得病毒能够吸附于细胞表面。病毒在受体的介导下最终进入细胞进行复制。细胞上的病毒受体与病毒的宿主范围和组织嗜性密切相关登革病毒是世界范围的重要的虫媒传染病尽管经历了几十年的研究,然而登革病毒的复制周期还是未知的。登革病毒可以在多种昆虫和哺乳动物细胞培养中增殖,并引起培养细胞发生折光性增强、细胞变圆或细胞融合等不同程度的细胞病变。在哺乳类细胞中的研究报道了大量细胞表面的登革病毒受体,其与细胞的种类和病毒的类型有关。哺乳动物的受体蛋白包括：细胞连接分子DC-SIGN(凝集素C受体),37/67kDa高亲和层粘连蛋白受体,及热休克蛋白HSP70/90。然而,昆虫细胞上的登革病毒受体并没有鉴定,主要的鉴定仅仅限制在分子量的大小上。然而目前大部分研究都没有区别是病毒连接分子还是作用分子,对于筛选出的分子与登革病毒的作用功能研究甚少。 虫媒媒介效能与产生媒介感染的媒介屏障有关,主要包括中肠感染屏障(midgut infection barrier, MIB)、中肠逃逸屏障(midgut escape barrier, MEB)、唾液腺播散屏障(salivary glands transmission barrier)。如果存在MIB,病毒就不能在蚊虫中肠细胞中感染或复制,这可能是由于细胞表面缺乏病毒受体。而MEB的存在,使病毒只能在中肠复制而不能播散,进一步感染二级靶器官。迄今为止,蚊传黄病毒与蚊虫中肠上皮细胞受体的相互作用机制还不十分清楚。本文在研究登革病毒受体的初选时,选用蚊虫的中肠组织作为主要研究对象,也是基于此媒介屏障的理论。 在选用C6/36细胞作为潜在登革病毒受体的筛选的载体,研究甚多,研究者利用VOPBA (virus overlay protein binding assay)病毒覆盖蛋白结合试验,将提取的C6/36细胞膜蛋白通过SDS-PAGE分离,转到固体膜上,再将纯化的登革病毒覆盖结合蛋白,通过针对登革病毒的单抗识别结合的病毒间接筛选出特异性蛋白条带。该方法早期由Mexico等应用在白纹伊蚊系C6/36细胞中筛选登革Ⅱ型病毒潜在受体,筛选出主要的分子为分子量约67000和80000的多肽。这为后来的登革受体研究在方法学上做了初步的探索,至今研究筛选受体分子应用VOPBA作为初步筛选是较为理想的方法,国内外研究者外对此方法也不断改进,在登革病毒受体研究上取得很大进展,用上述方法Munoz等在C6/36细胞中鉴定出分子量67kDa和80kDa的两种蛋白为DV2的受体蛋白,尤其是分子量为67kDa的蛋白其多克隆抗体够抑制DV2连接感染C6/36。之后的研究建议把这两种蛋白作为四型登革病毒的受体蛋白。Chee等用VOPBA的方法筛选出分子量为48kDa的分子,之后第一次用质谱分析的方法鉴定出该分子为微管样蛋白,微管蛋白在最初并不参与病毒内化作用,而是在后阶段的内化或是在搬运病毒颗粒发挥作用。在早期的研究中Salas-Benito等在C6/36细胞膜蛋白中鉴定出两种蛋白40kDa/45kDa,作为DV4的连接分子。并用亲和柱色谱法鉴定出这两种蛋白与重组登革病毒E蛋白相互作用,最终证明这两种蛋白为糖蛋白,尽管碳水化合物部分并未参与连接作用,之后的研究建议将该蛋白作为热休克蛋白(Hsp)90。通过抗体介导和小片段RNA介导的干扰下调抑制素基因的方法来确定抑制素介导病毒进入昆虫细胞的作用。发现抑制素的作用只针对登革Ⅱ型病毒,而不参与介导与日本脑炎病毒进入细胞。Paingankar等用VOPBA的方法在C6/36细胞、埃及伊蚊A7细胞及白纹伊蚊的中肠缘状膜组织中鉴定了七种连接蛋白(肌动蛋白、ATP合酶p亚组、热休克蛋白同系物70、orisis、微管蛋白p链、vav-1及抑制素prohibitin)它们共同协助病毒内化和转运。在该模型中,作者建议登革病毒和细胞的初始作用是以非蛋白—蛋白相互作用发生的包括层粘连蛋白,凝集素,硫酸乙酰肝素或类似的分子,继而由微管蛋白介导的病毒内化。 研究受体介导的登革病毒传播周期还处在探索阶段,至今并未有明确的一套机制定义该途径的作用分子,对于受体的研究一直以来都是热点,为今后药物靶点筛选的研究具有一定参考价值。 目的： 1.应用病毒覆盖结合蛋白实验(VOPBA),初步筛选C6/36细胞膜上与登革病毒作用的连接分子。 2.应用病毒覆盖结合蛋白实验(VOPBA),初步筛选白纹伊蚊中肠组织膜蛋白中与登革病毒作用的连接分子。 3.应用病毒覆盖结合蛋白实验(VOPBA),比较白纹伊蚊马氏管组织、卵巢组织、中肠组织与致倦库蚊中肠组织中登革病毒的连接分子,试图找出差异性结果解释媒介屏障作用。 4.选用二维电泳分离技术结合VOPBA,筛选特异结合分子,应用质谱分析技术分析特异蛋白,为该蛋白的功能研究做好铺垫。 5.设计埃及伊蚊的阴离子通道蛋白和埃及伊蚊核糖体蛋白S7的real-time PCR引物。 方法： 1.实验室条件下饲养白纹伊蚊、致倦库蚊、埃及伊蚊,温度(26±1)℃,相对湿度70%,光照周期12h：12h(光照：暗室)。用干酵母片饲养蚊幼虫,定时更换清水,保持水体清洁；成蚊每日饲喂含10%葡萄水,雌蚊在交配后(一般在羽化后三天)可供应小白鼠血餐1次,为产卵提供必需营养。 2.C6/36细胞的培养,28℃,于昆虫细胞孵箱中培养。 3.昆明乳鼠脑内注射接种登革病毒,待乳鼠出现发病症状,收集鼠脑研磨得到含病毒的上清,经C6/36细胞传代培养获得滴度更高的病毒液。 4.病毒的滴度测定采用单层细胞滴定法测定TCID50,即半数细胞培养物出现病变的最高稀释度,采用Reed-Muench法计算TCID50。 5.参考文献提取C6/36细胞、白纹伊蚊中肠组织、白纹伊蚊马氏管、白纹伊蚊卵巢、致倦库蚊中肠组织的膜蛋白。通过SDS-PAGE分离蛋白,结和病毒结合实验(VOPBA)初筛特异性结合分子。 6.应用二维电泳仪分离C6/36细胞膜蛋白,结合病毒结合试验(VOPBA)初筛特异性结合分子,质谱分析该特异蛋白的氨基酸序列。 7.利用DNAMAN和PrimerPremier5.0软件设计埃及伊蚊阴离子通道蛋白基因埃及伊蚊核糖体蛋白S7的qPCR引物,遵照qPCR引物原则。 8.Trizol法提取埃及伊蚊RNA,利用Takara公司RT-PCR试剂盒扩增目的基因。 结果： 1.登革病毒在白纹伊蚊系细胞C6/36细胞中成功复制,检测登革病毒滴为107.80TCID50/0.1mL 2.参考文献提取C6/36细胞、白纹伊蚊中肠、白纹伊蚊马氏管、白纹伊蚊卵巢,致倦库蚊中肠组织膜蛋白经SDS-PAGE蛋白胶分离显示蛋白条带清晰无降解,可以用于下游实验。 3.C6/36细胞膜蛋白病毒孵育组在30kDa有特异性条带,而阴性对照组无此条带。 4.研究蚊虫组织病毒结合试验中发现在白纹伊蚊中肠组织、白纹伊蚊马氏管带组织、白纹伊蚊卵巢组织,致倦库蚊中肠组织都在30kDa有结合条带。 5.质谱分析结果比对该蛋白与埃及伊蚊离子通道蛋白的匹配上的氨基酸为29%。 6.应用生物信息学分析,埃及伊蚊阴离子通道蛋白为非跨膜蛋白。 7.成功扩增埃及伊蚊离子通道蛋白和埃及伊蚊线粒体蛋白S7基因,长度分别为125bp和247bp,将测序结果比对原序列确定为目的基因。 结论： 1.应用VOPBA法在C6/36细胞中筛选出与登革Ⅱ型病毒连接分子,分子量大小为30kDa。 2.应用VOPBA法在白纹伊蚊中肠组织、白纹伊蚊马氏管组织、白纹伊蚊卵巢组织,致倦库蚊中肠组织都在30kDa都有结合条带。 3.质谱分析该结合蛋白与埃及伊蚊离子通道蛋白相似性高,初步鉴定该蛋白为离子通道蛋白,进一步对该蛋白进行研究。 4.生物信息学分析该蛋白的性质发现该蛋白为非跨膜蛋白,其参与的作用是连接还是作为伴侣分子有待研究。
[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锛，

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