本文關鍵詞： 惡性瘧原蟲 棒狀體蛋白3 細胞侵入 功能　出處：《吉林大學》2015年碩士論文　論文類型：學位論文

【摘要】：瘧疾是全球致死率最高的傳染病之一，有超過全世界一半的人口都在感染瘧疾的危險之下。在感染人的五種瘧原蟲中尤其以惡性瘧原蟲的危害最為嚴重，為了根除這種疾病人們采取了不同的措施，其中研制安全高效的疫苗是目前控制瘧疾流行最有效的手段之一，而瘧原蟲裂殖子表面蛋白和一些棒狀體、微線體蛋白在裂殖子侵染紅細胞時直接暴露在機體的免疫系統(tǒng)之下，因此被認為是瘧疾疫苗的候選因子。 瘧原蟲的裂殖子侵染紅細胞是一個復雜的和多步驟的過程，有越來越多的證據表明來自于瘧原蟲頂端細胞器（棒狀體和微線體）分泌的蛋白在這個過程中發(fā)揮了至關重要的作用。最近的研究結果顯示，在惡性瘧原蟲中三個棒狀體的頸部蛋白（PfRON2,4和5）與微線體分泌的頂端膜蛋白PfAMA1已經被證實了位于緊密連接處并會形成RONs-AMA1復合物并在裂殖子侵入紅細胞的過程中發(fā)揮了關鍵的作用，但是目前對于棒狀體蛋白3（PfRON3）的研究結果顯示其并沒有參與RONS-AMA1復合物的形成，而是形成了一個新的棒狀體復合物（PfRON2,3和4）來發(fā)揮作用，，但是對于PfRON3在裂殖子侵入紅細胞過程中的作用機制仍知之甚少。 本研究先是通過熒光定量PCR的方法檢測了PfRON3基因在蟲體發(fā)育繁殖過程中的表達規(guī)律；通過構建PfRON3的原核表達質粒，在大腸桿菌表達系統(tǒng)中誘導表達了PfRON3的重組蛋白，通過Western-blot，間接免疫熒光（IFA）和免疫電鏡對PfRON3的表達和定位情況進行了研究，使用重組蛋白對PfRON3與多糖類物質肝素的結合進行了分析，通過間接ELISA方法分析了瘧疾高發(fā)地區(qū)瘧疾患者血清對重組PfRON3蛋白的識別情況，同時使用重組蛋白與蟲體天然蛋白質對PfRON3與紅細胞的結合進行了研究，最后用抗PfRON3的特異性抗體進行了體外抑制蟲體侵染的實驗。 結果表明了PfRON3基因主要表達于惡性瘧原蟲侵入紅細胞后的裂殖體期并且在侵入紅細胞后在40h達到最高峰，而亞細胞結構定位結果顯示PfRON3定位在棒狀體的泡部，PfRON3的重組蛋白與蟲體天然蛋白均可以結合紅細胞說明了PfRON3含有一段區(qū)域可以介導裂殖子與紅細胞之間的聯(lián)系，并且PfRON3還可以和多糖類物質肝素進行特異性結合，說明了PfRON3很有可能通過紅細胞表面多糖類分子結合介導蟲體對紅細胞的侵入。PfRON3重組蛋白可以被非洲瘧疾高發(fā)地區(qū)瘧疾患者的血清識別并且抗PfRON3特異性抗體能夠有效地抑制蟲體侵入紅細胞說明了PfRON3在侵入的過程中是作為一個重要的免疫原存在的。 總之通過本研究得到的結論是：PfRON3是惡性瘧原蟲的一種重要功能蛋白質和潛在的瘧疾疫苗候選抗原。
[Abstract]:Malaria is one of the most deadly infectious diseases in the world, and more than half of the world's population is at risk of contracting malaria. Different measures have been taken to eradicate the disease, among which the development of a safe and efficient vaccine is one of the most effective means of controlling the malaria epidemic at present, while the parasite merozoite surface protein and some coryliform bodies, Microline proteins are directly exposed to the immune system when merozoites infect red blood cells, so they are considered as candidate factors for malaria vaccine. The infection of the merozoites of Plasmodium falciparum with red blood cells is a complex and multistep process. There is growing evidence that proteins secreted from the parasite's apical organelles (rods and microstrands) play a crucial role in this process. In Plasmodium falciparum, PfRON2O4 and 5) the apical membrane protein PfAMA1 secreted by the microline has been confirmed to be located at a tight junction and to form RONs-AMA1 complexes and play a key role in merozoites invading red blood cells. However, current studies on rodlike protein 3 (PfRON3) show that it does not participate in the formation of RONS-AMA1 complexes, but rather forms a new rodlike complex PfRON2O3 and 4) to play its role. However, little is known about the role of PfRON3 in the process of merozoite invasion into erythrocytes. In this study, the expression of PfRON3 gene was detected by fluorescence quantitative PCR, and the recombinant protein of PfRON3 was induced in Escherichia coli by constructing prokaryotic expression plasmid of PfRON3. The expression and localization of PfRON3 were studied by Western-blot, indirect immunofluorescence assay (IFA) and immunoelectron microscopy. The binding of PfRON3 to heparin was analyzed by recombinant protein. The recognition of recombinant PfRON3 protein in serum of malaria patients in high incidence areas was analyzed by indirect ELISA method, and the binding of PfRON3 to red blood cells was studied by using recombinant protein and natural protein of insect body. Finally, the specific antibody against PfRON3 was used to inhibit the infection of insect in vitro. The results showed that PfRON3 gene was mainly expressed in the merozoite phase after Plasmodium falciparum invaded red blood cells and reached its peak at 40 hours after invasion. The results of subcellular structural localization showed that the recombinant protein PfRON3 located in the rodlike vesicles and the natural proteins of the parasite could bind to the red blood cells, indicating that PfRON3 contains a region that mediates the connection between merozoites and erythrocytes. And PfRON3 can also specifically bind to heparin, a polysaccharide substance, It is suggested that PfRON3 may be mediated by polycarbohydrate molecule binding on erythrocyte surface. PfRON3 recombinant protein can be recognized by the sera of malaria patients in malaria-prone areas in Africa, and the specific antibody against PfRON3 can be obtained. The inhibitory effect of PfRON3 on the invasion of erythrocytes suggests that PfRON3 exists as an important immunogen during the process of invasion. It is concluded that: PfRON3 is an important functional protein of Plasmodium falciparum and a potential candidate antigen for malaria vaccine.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.7

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