本文選題：海倫微孢子蟲 + 極管蛋白�。� 參考：《西南大學(xué)》2017年博士論文

【摘要】：微孢子蟲是一種寄生于細(xì)胞內(nèi)的機(jī)會(huì)性病原,宿主廣泛,可通過(guò)食物和水進(jìn)行傳播,并引發(fā)微孢子蟲病。微孢子蟲具有獨(dú)特的侵染裝置——極管。極管從孢子彈出的過(guò)程稱為發(fā)芽,在孢子發(fā)芽前,極管在孢子內(nèi)纏繞于孢原質(zhì)的外周,在適當(dāng)?shù)耐饨绛h(huán)境刺激下,極管快速地彈出并刺穿細(xì)胞膜,將含有細(xì)胞核的侵染性孢原質(zhì)注入到宿主細(xì)胞。盡管對(duì)于極管形態(tài)結(jié)構(gòu)的描述和研究已經(jīng)有100多年,但目前仍然缺乏對(duì)極管組成機(jī)制、細(xì)胞黏附和侵染機(jī)制的深刻認(rèn)識(shí)。在對(duì)于微孢子蟲的極管蛋白研究中發(fā)現(xiàn)某些極管蛋白具有O-甘露糖基化修飾,可能與宿主細(xì)胞表面的甘露糖結(jié)合受體互作,在侵染過(guò)程中起作用。關(guān)于極管蛋白的構(gòu)成,目前的研究發(fā)現(xiàn),極管至少含有5種蛋白,其中已經(jīng)有三種蛋白被鑒定并報(bào)道。本論文中以前期研究得到的假定極管蛋白4(Eh PTP4)作為研究對(duì)象,聚焦該蛋白的鑒定、功能及其宿主細(xì)胞表面結(jié)合受體等科學(xué)問(wèn)題,從海倫微孢子蟲中得到的假定極管蛋白4的基因序列出發(fā),對(duì)其進(jìn)行了功能域以及糖基化修飾等序列信息的分析、重組蛋白的表達(dá)、單克隆抗體和多克隆抗體的制備以及免疫熒光電鏡定位分析,并利用流式細(xì)胞實(shí)驗(yàn)等對(duì)Eh PTP4的細(xì)胞學(xué)功能進(jìn)行了分析,利用免疫共沉淀的方法對(duì)其細(xì)胞受體蛋白進(jìn)行了鑒定,最后對(duì)Eh PTP4與其受體蛋白的互作關(guān)系及其在微孢子蟲侵染過(guò)程中的作用進(jìn)行了分析。本論文的主要結(jié)果總結(jié)為以下四個(gè)方面:1.海倫微孢子蟲Ehhptp4基因序列的生物信息學(xué)分析利用序列分析軟件和在線網(wǎng)站對(duì)Ehhptp4的基因序列特征進(jìn)行了分析發(fā)現(xiàn),Ehhptp4序列中含有一個(gè)信號(hào)肽,信號(hào)肽區(qū)域的氨基酸疏水性較高;Ehhptp4序列內(nèi)部存在一個(gè)幾丁質(zhì)結(jié)合基序,該結(jié)合基序中三個(gè)功能相關(guān)的氨基酸(半胱氨酸Cys、脯氨酸Pro以及甘氨酸Gly)之中,有兩個(gè)氨基酸(Cys和Gly)在Eh HPTP4內(nèi)具有保守性,表明Eh HPTP4可能具有幾丁質(zhì)結(jié)合功能,這是在微孢子蟲極管蛋白中首次發(fā)現(xiàn)的具有潛在幾丁質(zhì)結(jié)合能力的極管蛋白。另外,Eh HPTP4的C端尾部還包含1個(gè)富含組氨酸的基序(Histidine-rich region,HRR),可能在細(xì)胞黏附的過(guò)程中起著重要的作用。同時(shí),在Eh HPTP4中含有6個(gè)半胱氨酸,其中4個(gè)在多種微孢子蟲PTP4蛋白序列中高度保守,推測(cè)其在PTP4與其它蛋白互作的過(guò)程中起作用。另外,糖基化以及磷酸化位點(diǎn)預(yù)測(cè)顯示,Eh HPTP4存在不同程度的糖基化修飾和磷酸化修飾,表明該蛋白可能屬于糖蛋白,在細(xì)胞黏附的過(guò)程中發(fā)揮功能。另外,亞細(xì)胞定位預(yù)測(cè)顯示Eh HPTP4是分泌型蛋白。2.海倫微孢子蟲Eh HPTP4的定位分析通過(guò)對(duì)Eh HPTP4進(jìn)行原核蛋白表達(dá)以及純化,制備了相應(yīng)單克隆和多克隆抗體。免疫印跡實(shí)驗(yàn)結(jié)果表明,制備的抗體可以清晰地與原核表達(dá)的Eh HPTP4以及孢子總蛋白中的Eh HPTP4發(fā)生免疫反應(yīng);間接免疫熒光實(shí)驗(yàn)、免疫透射電鏡實(shí)驗(yàn)以及光電聯(lián)合顯微鏡技術(shù)分析發(fā)現(xiàn),兔多克隆抗體可以將Eh HPTP4定位于極管的全域,表明Eh HPTP4為極管蛋白的新成員,將其命名為極管蛋白4(Eh PTP4)。而Eh PTP4單克隆抗體卻只能標(biāo)記彈出后極管的最前端,該結(jié)果表明,在極管蛋白合成以及極管組裝的過(guò)程中,Eh PTP4在極管不同部位可能存在不同的蛋白構(gòu)象,各自行使不同的功能。Eh PTP4特殊抗原表位暴露于極管最前端,可能在極管與宿主細(xì)胞互作等方面發(fā)揮重要功能。3.海倫微孢子蟲EhPTP4的功能分析基于序列信息分析發(fā)現(xiàn),Eh PTP4具有幾丁質(zhì)結(jié)合功能,通過(guò)真核表達(dá)獲得Eh PTP4的重組蛋白,利用堿溶法提取了微孢子蟲脫蛋白幾丁質(zhì)殼(DCSCs),并利用IFA實(shí)驗(yàn)等證明了重組Eh PTP4蛋白以及天然Eh PTP4蛋白具有幾丁質(zhì)結(jié)合功能,表明Eh PTP4對(duì)極管在孢子內(nèi)的固定、或者與宿主細(xì)胞表面的糖蛋白結(jié)合有關(guān)。利用免疫印跡實(shí)驗(yàn)、非變性凝膠電泳實(shí)驗(yàn)以及免疫共沉淀實(shí)驗(yàn)等對(duì)Eh PTP4多克隆抗體和單克隆抗體的免疫原性進(jìn)行分析,結(jié)果表明,雖然兩種抗體具有不同的定位特征,但是它們均可識(shí)別孢子總蛋白中同一個(gè)蛋白,即Eh PTP4,表明兩種抗體的免疫原性都來(lái)自于Eh PTP4上的抗原表位,而單克隆抗體不能識(shí)別形成復(fù)合體的Eh PTP4,表明極管在組裝過(guò)程中有部分Eh PTP4在極管頂端以單體的形式存在,而在極管其它區(qū)域則以聚合體的形式存在,參與極管的組裝過(guò)程。進(jìn)一步,本研究利用ELISA、IFA以及FACS等方法對(duì)Eh PTP4的細(xì)胞結(jié)合功能進(jìn)行了分析,結(jié)果表明Eh PTP4具有宿主細(xì)胞結(jié)合功能,可與宿主細(xì)胞表面的某些未知成分互作,暗示其可能在極管侵染宿主的過(guò)程中起著重要的作用。4.海倫微孢子蟲Eh PTP4細(xì)胞受體的鑒定以及分析在微孢子蟲侵染機(jī)制研究中,對(duì)其侵染相關(guān)的細(xì)胞受體的研究未見任何報(bào)道,處于空白狀態(tài)。本研究在Eh PTP4具有細(xì)胞結(jié)合能力的基礎(chǔ)上,首先通過(guò)免疫共沉淀結(jié)合蛋白組學(xué)分析的方法,對(duì)其細(xì)胞受體進(jìn)行了篩查,發(fā)現(xiàn)位于細(xì)胞表面的膜蛋白轉(zhuǎn)鐵蛋白受體1(Transferrin receptor 1,Tf R-1)為其潛在的細(xì)胞受體之一。為了進(jìn)一步證明這二者之間的互作關(guān)系,分別通過(guò)pull down實(shí)驗(yàn)、非變性凝膠電泳實(shí)驗(yàn)以及熒光共定位實(shí)驗(yàn)等,對(duì)Eh PTP4與Tf R-1的互作關(guān)系進(jìn)行分析驗(yàn)證,結(jié)果證明了兩者間具有相互作用,表明這兩個(gè)蛋白在極管進(jìn)入宿主細(xì)胞的過(guò)程中,能夠借助彼此之間的相互作用促進(jìn)微孢子蟲入侵宿主細(xì)胞。為了進(jìn)一步證明Eh PTP4和Tf R-1在微孢子蟲侵染宿主過(guò)程中所起的作用,分別利用抗體封閉實(shí)驗(yàn),將原核表達(dá)蛋白加入細(xì)胞的培養(yǎng)體系,以及從宿主細(xì)胞上敲除轉(zhuǎn)鐵蛋白受體等方法,證明了當(dāng)Eh PTP4與Tf R-1之間的互作受到影響時(shí),微孢子蟲對(duì)宿主的侵染能力也會(huì)受到很大程度的抑制,進(jìn)一步確認(rèn)了Eh PTP4以及Tf R-1在微孢子蟲侵染宿主過(guò)程中的重要性。另外,鑒于Tf R-1是通過(guò)受體蛋白介導(dǎo)的內(nèi)吞作用將鐵離子運(yùn)輸進(jìn)入細(xì)胞,利用小分子抑制劑Pitstop 2對(duì)內(nèi)吞作用進(jìn)行抑制,發(fā)現(xiàn)同時(shí)抑制了微孢子蟲對(duì)細(xì)胞的侵染,暗示微孢子蟲極管頂端在接觸到宿主細(xì)胞后,可能借助Eh PTP4和Tf R-1的互作,通過(guò)細(xì)胞內(nèi)吞的作用進(jìn)入細(xì)胞內(nèi)部。綜上所述,在本研究中,我們對(duì)海倫微孢子蟲極管蛋白4(EhPTP4)進(jìn)行了鑒定以及功能分析,并且首次發(fā)現(xiàn)了與微孢子蟲侵染相關(guān)的細(xì)胞受體蛋白Tf R-1,并分析證明了Eh PTP4和Tf R-1之間具有互作關(guān)系,同時(shí)對(duì)這種互作關(guān)系在微孢子蟲侵染宿主過(guò)程中的作用進(jìn)行了研究,證明了海倫微孢子蟲極管頂端是通過(guò)細(xì)胞內(nèi)吞的作用進(jìn)入宿主細(xì)胞以實(shí)現(xiàn)微孢子蟲的入侵感染。本論文的研究結(jié)果為微孢子蟲侵染機(jī)制的研究提供新的線索和思路,并為最終微孢子蟲機(jī)制的闡明奠定了強(qiáng)有力的基礎(chǔ)。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S884
，

本文編號(hào)：2082606