海伦微孢子虫极管蛋白EhPTP4的鉴定及其在侵染宿主细胞过程中的作用研究

发布时间：2018-06-29 15:48

  本文选题：海伦微孢子虫 + 极管蛋白　； 参考：《西南大学》2017年博士论文

【摘要】：微孢子虫是一种寄生于细胞内的机会性病原,宿主广泛,可通过食物和水进行传播,并引发微孢子虫病。微孢子虫具有独特的侵染装置——极管。极管从孢子弹出的过程称为发芽,在孢子发芽前,极管在孢子内缠绕于孢原质的外周,在适当的外界环境刺激下,极管快速地弹出并刺穿细胞膜,将含有细胞核的侵染性孢原质注入到宿主细胞。尽管对于极管形态结构的描述和研究已经有100多年,但目前仍然缺乏对极管组成机制、细胞黏附和侵染机制的深刻认识。在对于微孢子虫的极管蛋白研究中发现某些极管蛋白具有O-甘露糖基化修饰,可能与宿主细胞表面的甘露糖结合受体互作,在侵染过程中起作用。关于极管蛋白的构成,目前的研究发现,极管至少含有5种蛋白,其中已经有三种蛋白被鉴定并报道。本论文中以前期研究得到的假定极管蛋白4(Eh PTP4)作为研究对象,聚焦该蛋白的鉴定、功能及其宿主细胞表面结合受体等科学问题,从海伦微孢子虫中得到的假定极管蛋白4的基因序列出发,对其进行了功能域以及糖基化修饰等序列信息的分析、重组蛋白的表达、单克隆抗体和多克隆抗体的制备以及免疫荧光电镜定位分析,并利用流式细胞实验等对Eh PTP4的细胞学功能进行了分析,利用免疫共沉淀的方法对其细胞受体蛋白进行了鉴定,最后对Eh PTP4与其受体蛋白的互作关系及其在微孢子虫侵染过程中的作用进行了分析。本论文的主要结果总结为以下四个方面:1.海伦微孢子虫Ehhptp4基因序列的生物信息学分析利用序列分析软件和在线网站对Ehhptp4的基因序列特征进行了分析发现,Ehhptp4序列中含有一个信号肽,信号肽区域的氨基酸疏水性较高;Ehhptp4序列内部存在一个几丁质结合基序,该结合基序中三个功能相关的氨基酸(半胱氨酸Cys、脯氨酸Pro以及甘氨酸Gly)之中,有两个氨基酸(Cys和Gly)在Eh HPTP4内具有保守性,表明Eh HPTP4可能具有几丁质结合功能,这是在微孢子虫极管蛋白中首次发现的具有潜在几丁质结合能力的极管蛋白。另外,Eh HPTP4的C端尾部还包含1个富含组氨酸的基序(Histidine-rich region,HRR),可能在细胞黏附的过程中起着重要的作用。同时,在Eh HPTP4中含有6个半胱氨酸,其中4个在多种微孢子虫PTP4蛋白序列中高度保守,推测其在PTP4与其它蛋白互作的过程中起作用。另外,糖基化以及磷酸化位点预测显示,Eh HPTP4存在不同程度的糖基化修饰和磷酸化修饰,表明该蛋白可能属于糖蛋白,在细胞黏附的过程中发挥功能。另外,亚细胞定位预测显示Eh HPTP4是分泌型蛋白。2.海伦微孢子虫Eh HPTP4的定位分析通过对Eh HPTP4进行原核蛋白表达以及纯化,制备了相应单克隆和多克隆抗体。免疫印迹实验结果表明,制备的抗体可以清晰地与原核表达的Eh HPTP4以及孢子总蛋白中的Eh HPTP4发生免疫反应;间接免疫荧光实验、免疫透射电镜实验以及光电联合显微镜技术分析发现,兔多克隆抗体可以将Eh HPTP4定位于极管的全域,表明Eh HPTP4为极管蛋白的新成员,将其命名为极管蛋白4(Eh PTP4)。而Eh PTP4单克隆抗体却只能标记弹出后极管的最前端,该结果表明,在极管蛋白合成以及极管组装的过程中,Eh PTP4在极管不同部位可能存在不同的蛋白构象,各自行使不同的功能。Eh PTP4特殊抗原表位暴露于极管最前端,可能在极管与宿主细胞互作等方面发挥重要功能。3.海伦微孢子虫EhPTP4的功能分析基于序列信息分析发现,Eh PTP4具有几丁质结合功能,通过真核表达获得Eh PTP4的重组蛋白,利用碱溶法提取了微孢子虫脱蛋白几丁质壳(DCSCs),并利用IFA实验等证明了重组Eh PTP4蛋白以及天然Eh PTP4蛋白具有几丁质结合功能,表明Eh PTP4对极管在孢子内的固定、或者与宿主细胞表面的糖蛋白结合有关。利用免疫印迹实验、非变性凝胶电泳实验以及免疫共沉淀实验等对Eh PTP4多克隆抗体和单克隆抗体的免疫原性进行分析,结果表明,虽然两种抗体具有不同的定位特征,但是它们均可识别孢子总蛋白中同一个蛋白,即Eh PTP4,表明两种抗体的免疫原性都来自于Eh PTP4上的抗原表位,而单克隆抗体不能识别形成复合体的Eh PTP4,表明极管在组装过程中有部分Eh PTP4在极管顶端以单体的形式存在,而在极管其它区域则以聚合体的形式存在,参与极管的组装过程。进一步,本研究利用ELISA、IFA以及FACS等方法对Eh PTP4的细胞结合功能进行了分析,结果表明Eh PTP4具有宿主细胞结合功能,可与宿主细胞表面的某些未知成分互作,暗示其可能在极管侵染宿主的过程中起着重要的作用。4.海伦微孢子虫Eh PTP4细胞受体的鉴定以及分析在微孢子虫侵染机制研究中,对其侵染相关的细胞受体的研究未见任何报道,处于空白状态。本研究在Eh PTP4具有细胞结合能力的基础上,首先通过免疫共沉淀结合蛋白组学分析的方法,对其细胞受体进行了筛查,发现位于细胞表面的膜蛋白转铁蛋白受体1(Transferrin receptor 1,Tf R-1)为其潜在的细胞受体之一。为了进一步证明这二者之间的互作关系,分别通过pull down实验、非变性凝胶电泳实验以及荧光共定位实验等,对Eh PTP4与Tf R-1的互作关系进行分析验证,结果证明了两者间具有相互作用,表明这两个蛋白在极管进入宿主细胞的过程中,能够借助彼此之间的相互作用促进微孢子虫入侵宿主细胞。为了进一步证明Eh PTP4和Tf R-1在微孢子虫侵染宿主过程中所起的作用,分别利用抗体封闭实验,将原核表达蛋白加入细胞的培养体系,以及从宿主细胞上敲除转铁蛋白受体等方法,证明了当Eh PTP4与Tf R-1之间的互作受到影响时,微孢子虫对宿主的侵染能力也会受到很大程度的抑制,进一步确认了Eh PTP4以及Tf R-1在微孢子虫侵染宿主过程中的重要性。另外,鉴于Tf R-1是通过受体蛋白介导的内吞作用将铁离子运输进入细胞,利用小分子抑制剂Pitstop 2对内吞作用进行抑制,发现同时抑制了微孢子虫对细胞的侵染,暗示微孢子虫极管顶端在接触到宿主细胞后,可能借助Eh PTP4和Tf R-1的互作,通过细胞内吞的作用进入细胞内部。综上所述,在本研究中,我们对海伦微孢子虫极管蛋白4(EhPTP4)进行了鉴定以及功能分析,并且首次发现了与微孢子虫侵染相关的细胞受体蛋白Tf R-1,并分析证明了Eh PTP4和Tf R-1之间具有互作关系,同时对这种互作关系在微孢子虫侵染宿主过程中的作用进行了研究,证明了海伦微孢子虫极管顶端是通过细胞内吞的作用进入宿主细胞以实现微孢子虫的入侵感染。本论文的研究结果为微孢子虫侵染机制的研究提供新的线索和思路,并为最终微孢子虫机制的阐明奠定了强有力的基础。
[Abstract]:Microspore is an opportunistic pathogen that is parasitic in cells. The host is widely distributed and can be transmitted through food and water and triggering microspore disease. The microspore insect has a unique infection device - the polar tube. The process of the pole tube from spores is called germination. The tube is entangled in the sporosporium in the spore before the spore germinates and is appropriate. Under the external environment, the polar tube quickly popped out and pierced the cell membrane and injected the infected sporogenesis containing the nucleus into the host cell. Although the morphological structure of the polar tube had been described and studied for more than 100 years, there was still a lack of deep understanding of the mechanism of polar tube composition, cell adhesion and infection mechanism. In the study of the polar tubulin, some polar tubulin have been found to have O- mannose based modification, which may interact with mannose binding receptors on the surface of the host cell and play a role in the process of infection. On the composition of the polar tubulin, the current research has found that at least 5 kinds of proteins have been contained in the polar tube, of which three proteins have been identified and reported. In this paper, the presumed polar tube protein 4 (Eh PTP4), as an object of study, focused on the identification of the protein, its function and its host cell surface binding receptor and other scientific problems, and the sequence information of functional domain and glycosylation modification from the gene sequence of the presumed tube protein 4 obtained from the microspore of Helen's microspporia. The expression of recombinant protein, the preparation of monoclonal antibody and polyclonal antibody, the localization and analysis of immunofluorescence electron microscopy, and the analysis of the cytological function of Eh PTP4 by flow cytometry, and the identification of the cell receptor protein by the method of CO immunoprecipitation, and the interaction between Eh PTP4 and its receptor protein. The main results of this paper are summarized in the following four aspects: 1. the bioinformatics analysis of the Ehhptp4 gene sequence of microspore Helen, the sequence analysis software and the online website, and the analysis of the gene sequence characteristics of Ehhptp4, there is one in the Ehhptp4 sequence. Signal peptide, the amino acid hydrophobicity of the signal peptide region is high; there is a chitin binding sequence within the Ehhptp4 sequence, and the three functional amino acids (cysteine Cys, proline Pro and glycine Gly) in the binding sequence, two amino acids (Cys and Gly) have conservatism in Eh HPTP4, indicating that Eh HPTP4 may have a few In addition, the C end of Eh HPTP4 also contains 1 histidine rich sequences (Histidine-rich region, HRR), which may play an important role in cell adhesion. At the same time, there are 6 in Eh HPTP4. Cysteine, 4 of them highly conserved in the PTP4 protein sequence of a variety of microspore, speculated that it played a role in the interaction between PTP4 and other proteins. In addition, glycosylation and phosphorylation sites predicted that Eh HPTP4 had different degrees of glycosylated and phosphorylated modification, indicating that the protein might belong to glycoprotein and in cell adhesion. In addition, the subcellular localization prediction showed that Eh HPTP4 was the secretory protein.2. Helen microspore Eh HPTP4, which was expressed and purified by the expression and purification of the prokaryotic protein of Eh HPTP4, and the corresponding monoclonal and polyclonal antibody were prepared. The immunoblotting experiment results showed that the antibody prepared could be clearly with the prokaryotic cell. The expression of Eh HPTP4 and the immunoreaction of Eh HPTP4 in the spore total protein; indirect immunofluorescence, transmission electron microscopy and photoelectric combined microscopy analysis found that the rabbit polyclonal antibody can locate Eh HPTP4 in the whole domain of the polar tube, indicating that Eh HPTP4 is a new member of the polar tube protein, and is named as the polar tube protein 4 (Eh). PTP4). But the Eh PTP4 monoclonal antibody can only mark the most front-end of the pop-up pole tube. This result shows that in the process of polar tube protein synthesis and polar tube assembly, Eh PTP4 may have different protein conformations in different parts of the polar tube, each exerts a different function of.Eh PTP4 specific antigen epitopes exposed to the most front of the polar tube, possibly in the polar tube. The functional analysis of the host cell interaction, such as the interaction of.3. Helen microspore EhPTP4, shows that Eh PTP4 has a chitin binding function, and the recombinant protein of Eh PTP4 is obtained by eukaryotic expression. The protein chitin shell (DCSCs) of microspore microspore is extracted by alkali solution, and has been proved by IFA experiment. The recombinant Eh PTP4 protein and natural Eh PTP4 protein have chitin binding function, indicating that Eh PTP4 is fixed in the spores of the polar tube or associated with the glycoprotein binding on the surface of the host cell. The immunoblotting experiment, the non denaturing gel electrophoresis experiment and the immunoprecipitation experiment on the Eh PTP4 polyclonal and monoclonal antibodies The results of Phytophthora analysis showed that, although the two antibodies had different localization characteristics, they could identify the same protein in the total spore protein, Eh PTP4, indicating that the immunogenicity of the two antibodies came from the epitopes on the Eh PTP4, and the monoclonal antibodies could not identify the Eh PTP4 forming the complex, indicating that the polar tubes were assembled. In the process, some Eh PTP4 exists in the form of a single body at the tip of the polar tube, while the other regions in the polar tube exist in the form of polymer and participate in the assembly process of the polar tube. Further, this study analyzed the cell binding function of Eh PTP4 by means of ELISA, IFA and FACS. The results showed that Eh PTP4 had the host cell binding function, It can be interacted with some unknown components on the surface of the host cell, suggesting that it may play an important role in the process of infection of the host by the polar tube,.4. Helen microspore Eh PTP4 cell receptor identification and analysis in the study of microspporidium infection mechanism, the study of its infection related cell body has not been reported, in a blank state. In this study, on the basis of the cell binding ability of Eh PTP4, the cell receptor was screened by immunoprecipitation combined with proteomic analysis, and the membrane protein transferrin receptor 1 (Transferrin receptor 1, Tf R-1) located on the surface of the cell was one of its potential cell receptors. In order to further prove this two The interaction between the pull down experiments, the non denaturing gel electrophoresis and the fluorescence co localization experiment, respectively, is used to verify the interaction between Eh PTP4 and Tf R-1. The results show that the two proteins have interaction with each other, indicating that these proteins can be used in the process of entering the host cell in the polar tube, and can be used by the phase between each other. Interaction promotes microspore invasion of host cells. In order to further demonstrate the role of Eh PTP4 and Tf R-1 in the process of infecting host by microspore, antibody closure experiments were used to separate the prokaryotic expression proteins into the cell culture system and to knock out the transferrin receptor from the host cells, which proved that Eh PTP4 and Tf R were used. When the interaction between -1 is affected, the infection ability of microspore to host can also be suppressed to a great extent. It further confirms the importance of Eh PTP4 and Tf R-1 in the process of infecting host by microspore. In addition, Tf R-1 is used to transport iron ions into cells by the endocytosis of receptor protein and to use small molecules to inhibit it. Pitstop 2 inhibits endocytosis and inhibits the infection of microspore to cells at the same time, suggesting that the apex of the microspore tube may be interacted with Eh PTP4 and Tf R-1 to enter the cell through the interaction of endocytic endocytosis at the top of the microspore tube. In this study, we described the microspore microspore polar tube in this study. Protein 4 (EhPTP4) was identified and functional analysis, and the cell receptor protein Tf R-1 associated with microspore infection was discovered for the first time, and the interaction between Eh PTP4 and Tf R-1 was demonstrated. The interaction of this interaction in the host process of microspore infection was studied, and the microspore Helen microspore was proved. The tip of the polar tube enters the host cell through the endocytosis of the cell to realize the invasion and infection of microspore. The results of this paper provide new clues and ideas for the study of the mechanism of microspore infection, and lay a strong foundation for the elucidation of the mechanism of the final microspore worm.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S884
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本文编号：2082606