恶性疟原虫棒状体蛋白3的表达及功能分析

发布时间：2018-02-24 09:10

本文关键词： 恶性疟原虫棒状体蛋白3 细胞侵入功能　出处：《吉林大学》2015年硕士论文　论文类型：学位论文

【摘要】：疟疾是全球致死率最高的传染病之一，有超过全世界一半的人口都在感染疟疾的危险之下。在感染人的五种疟原虫中尤其以恶性疟原虫的危害最为严重，为了根除这种疾病人们采取了不同的措施，其中研制安全高效的疫苗是目前控制疟疾流行最有效的手段之一，而疟原虫裂殖子表面蛋白和一些棒状体、微线体蛋白在裂殖子侵染红细胞时直接暴露在机体的免疫系统之下，因此被认为是疟疾疫苗的候选因子。疟原虫的裂殖子侵染红细胞是一个复杂的和多步骤的过程，有越来越多的证据表明来自于疟原虫顶端细胞器（棒状体和微线体）分泌的蛋白在这个过程中发挥了至关重要的作用。最近的研究结果显示，在恶性疟原虫中三个棒状体的颈部蛋白（PfRON2,4和5）与微线体分泌的顶端膜蛋白PfAMA1已经被证实了位于紧密连接处并会形成RONs-AMA1复合物并在裂殖子侵入红细胞的过程中发挥了关键的作用，但是目前对于棒状体蛋白3（PfRON3）的研究结果显示其并没有参与RONS-AMA1复合物的形成，而是形成了一个新的棒状体复合物（PfRON2,3和4）来发挥作用，，但是对于PfRON3在裂殖子侵入红细胞过程中的作用机制仍知之甚少。本研究先是通过荧光定量PCR的方法检测了PfRON3基因在虫体发育繁殖过程中的表达规律；通过构建PfRON3的原核表达质粒，在大肠杆菌表达系统中诱导表达了PfRON3的重组蛋白，通过Western-blot，间接免疫荧光（IFA）和免疫电镜对PfRON3的表达和定位情况进行了研究，使用重组蛋白对PfRON3与多糖类物质肝素的结合进行了分析，通过间接ELISA方法分析了疟疾高发地区疟疾患者血清对重组PfRON3蛋白的识别情况，同时使用重组蛋白与虫体天然蛋白质对PfRON3与红细胞的结合进行了研究，最后用抗PfRON3的特异性抗体进行了体外抑制虫体侵染的实验。结果表明了PfRON3基因主要表达于恶性疟原虫侵入红细胞后的裂殖体期并且在侵入红细胞后在40h达到最高峰，而亚细胞结构定位结果显示PfRON3定位在棒状体的泡部，PfRON3的重组蛋白与虫体天然蛋白均可以结合红细胞说明了PfRON3含有一段区域可以介导裂殖子与红细胞之间的联系，并且PfRON3还可以和多糖类物质肝素进行特异性结合，说明了PfRON3很有可能通过红细胞表面多糖类分子结合介导虫体对红细胞的侵入。PfRON3重组蛋白可以被非洲疟疾高发地区疟疾患者的血清识别并且抗PfRON3特异性抗体能够有效地抑制虫体侵入红细胞说明了PfRON3在侵入的过程中是作为一个重要的免疫原存在的。总之通过本研究得到的结论是：PfRON3是恶性疟原虫的一种重要功能蛋白质和潜在的疟疾疫苗候选抗原。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7

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