發(fā)布時(shí)間：2018-02-24 09:10

  本文關(guān)鍵詞： 惡性瘧原蟲(chóng) 棒狀體蛋白3 細(xì)胞侵入 功能　出處：《吉林大學(xué)》2015年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：瘧疾是全球致死率最高的傳染病之一，有超過(guò)全世界一半的人口都在感染瘧疾的危險(xiǎn)之下。在感染人的五種瘧原蟲(chóng)中尤其以惡性瘧原蟲(chóng)的危害最為嚴(yán)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(chóng)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(xì)胞時(shí)直接暴露在機(jī)體的免疫系統(tǒng)之下，因此被認(rèn)為是瘧疾疫苗的候選因子。 瘧原蟲(chóng)的裂殖子侵染紅細(xì)胞是一個(gè)復(fù)雜的和多步驟的過(guò)程，有越來(lái)越多的證據(jù)表明來(lái)自于瘧原蟲(chóng)頂端細(xì)胞器（棒狀體和微線體）分泌的蛋白在這個(gè)過(guò)程中發(fā)揮了至關(guān)重要的作用。最近的研究結(jié)果顯示，在惡性瘧原蟲(chóng)中三個(gè)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(jīng)被證實(shí)了位于緊密連接處并會(huì)形成RONs-AMA1復(fù)合物并在裂殖子侵入紅細(xì)胞的過(guò)程中發(fā)揮了關(guān)鍵的作用，但是目前對(duì)于棒狀體蛋白3（PfRON3）的研究結(jié)果顯示其并沒(méi)有參與RONS-AMA1復(fù)合物的形成，而是形成了一個(gè)新的棒狀體復(fù)合物（PfRON2,3和4）來(lái)發(fā)揮作用，，但是對(duì)于PfRON3在裂殖子侵入紅細(xì)胞過(guò)程中的作用機(jī)制仍知之甚少。 本研究先是通過(guò)熒光定量PCR的方法檢測(cè)了PfRON3基因在蟲(chóng)體發(fā)育繁殖過(guò)程中的表達(dá)規(guī)律；通過(guò)構(gòu)建PfRON3的原核表達(dá)質(zhì)粒，在大腸桿菌表達(dá)系統(tǒng)中誘導(dǎo)表達(dá)了PfRON3的重組蛋白，通過(guò)Western-blot，間接免疫熒光（IFA）和免疫電鏡對(duì)PfRON3的表達(dá)和定位情況進(jìn)行了研究，使用重組蛋白對(duì)PfRON3與多糖類(lèi)物質(zhì)肝素的結(jié)合進(jìn)行了分析，通過(guò)間接ELISA方法分析了瘧疾高發(fā)地區(qū)瘧疾患者血清對(duì)重組PfRON3蛋白的識(shí)別情況，同時(shí)使用重組蛋白與蟲(chóng)體天然蛋白質(zhì)對(duì)PfRON3與紅細(xì)胞的結(jié)合進(jìn)行了研究，最后用抗PfRON3的特異性抗體進(jìn)行了體外抑制蟲(chóng)體侵染的實(shí)驗(yàn)。 結(jié)果表明了PfRON3基因主要表達(dá)于惡性瘧原蟲(chóng)侵入紅細(xì)胞后的裂殖體期并且在侵入紅細(xì)胞后在40h達(dá)到最高峰，而亞細(xì)胞結(jié)構(gòu)定位結(jié)果顯示PfRON3定位在棒狀體的泡部，PfRON3的重組蛋白與蟲(chóng)體天然蛋白均可以結(jié)合紅細(xì)胞說(shuō)明了PfRON3含有一段區(qū)域可以介導(dǎo)裂殖子與紅細(xì)胞之間的聯(lián)系，并且PfRON3還可以和多糖類(lèi)物質(zhì)肝素進(jìn)行特異性結(jié)合，說(shuō)明了PfRON3很有可能通過(guò)紅細(xì)胞表面多糖類(lèi)分子結(jié)合介導(dǎo)蟲(chóng)體對(duì)紅細(xì)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(shí)別并且抗PfRON3特異性抗體能夠有效地抑制蟲(chóng)體侵入紅細(xì)胞說(shuō)明了PfRON3在侵入的過(guò)程中是作為一個(gè)重要的免疫原存在的。 總之通過(guò)本研究得到的結(jié)論是：PfRON3是惡性瘧原蟲(chóng)的一種重要功能蛋白質(zhì)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(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S852.7

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