约氏疟原虫子孢子侵入肝细胞相关基因及蛋白研究

发布时间：2018-03-15 23:19

本文选题：约氏疟原虫　切入点：子孢子　出处：《第三军医大学》2005年硕士论文　论文类型：学位论文

【摘要】：蚊媒传播的疟疾是一种危害严重的热带传染病,在全世界人群中具有很高的发病率和致死率。近年来疟原虫多药抗性株的出现与迅速扩散,以及蚊媒对杀虫剂耐受性的增加,目前又无有效的抗疟疫苗,给疟疾防治工作带来很大的困难。传统方法已难于控制该病的流行,迫切需要为疟疾的防治工作提供新的方法和手段。疟疾是由蚊虫叮咬而传播,疟原虫子孢子一旦进入血循环,数分钟后便随血流跨过肝血窦枯否氏细胞(Kuppfer cell,KC)后主动粘附、迅速侵入肝细胞,并进行红外期增殖,然后才侵入红细胞,导致疟疾的发作。子孢子侵入肝细胞是疟疾感染的关键,而阻断虫体的侵入,即可防止疟疾感染,红外期阻断疫苗旨在阻断子孢子侵入宿主肝细胞,防止疟原虫红内期的发育,是疟疾疫苗的重要组成部分。唾液腺子孢子感染性受发育调节,正常情况下,中肠内卵囊成熟后破裂,子孢子逸出,经血淋巴移行到唾液腺后2 d,发育成为具有感染性的子孢子。因此研究疟原虫具有侵入肝细胞能力的唾液腺子孢子与不具侵入肝细胞能力的卵囊子孢子之间的差异分子具有重要意义。但现有的实验技术尚不支持从媒介蚊的中肠和唾液腺中分离获得高纯度、足量的子孢子,因此针对这个时期的研究工作存在较大难度。已经完成的恶性疟原虫、其媒介冈比亚按蚊和约氏疟原虫全基因组测序工作,以及其他种类疟原虫(间日疟原虫、伯氏疟原虫、诺氏疟原虫、鸡疟原虫)大量基因组序列的获得,为后续的疟原虫研究打下了坚实基础,基因功能的分析和蛋白质组学研究已成为后基因组时代的主要任务。通过对疟原虫子孢子肝细胞侵入相关的差异基因和蛋白的大量发现和鉴定,将有助于认识疟原虫生长发育的分子机制,可为疟疾的防治提供分子理论依据,提供药物和疫苗新的靶目标,为采取防治策略战胜疟疾打下了坚实的基础。因此本实验以斯氏按蚊-约氏疟原虫为模型,从基因和蛋白两方面对子孢子侵入肝细胞相关分子进行研究:一方面应用差异显示PCR (differential display RT-PCR, DD-PCR)技术筛选约氏疟原虫唾液腺和卵囊子孢子差异cDNA,经比较分析后获得唾液腺子孢子特有的差异序列,克隆至TA 载体,根据序列同源分析和功能预测结果,推测可能与子孢子侵入肝细胞相关的基因;另一方面采用二维凝胶电泳
[Abstract]:Mosquito-borne malaria is a highly harmful tropical infectious disease with high morbidity and mortality among people around the world. In recent years, the emergence and rapid spread of multidrug resistant strains of Plasmodium, as well as the increased resistance of mosquito vectors to insecticides, At present, there is no effective antimalarial vaccine, which poses great difficulties in malaria control. Traditional methods have become difficult to control the epidemic and there is an urgent need to provide new methods and means for malaria control. Malaria is transmitted by mosquito bites. Once the plasmodium sporozoites enter the blood circulation, after a few minutes with the blood flow across the hepatic sinusoidal Kuppfer cell Kuppfer cell (KCC), the plasmodium actively adheres, rapidly invades the hepatocyte, and carries on the infrared phase proliferation, then invades the red blood cell. It is the key of malaria infection that the sporozoite invades the liver cells, and blocking the invasion of the parasite can prevent the malaria infection. The infrared phase blocking vaccine is designed to prevent the sporozoite from invading the host liver cells and preventing the development of the erythroid phase of the malaria parasite. Salivary gland sporozoite infection is regulated by development. Normally, the oocysts in the midgut break down after maturation, and the sporozoites escape. Two days after the hemolymph migration to the salivary glands, they developed into infective sporozoites. Therefore, the difference between the salivary gland sporozoites with the ability to invade the liver cells and the oocystis with no ability to invade the hepatocytes was studied. Heteromolecules are of great significance, but existing experimental techniques do not support the isolation of high purity from the midgut and salivary glands of vector mosquitoes. Due to the sufficient number of sporozoites, it is difficult to do research on this period. The complete genome sequencing of Plasmodium falciparum, the vector Anopheles gambiae and Plasmodium yoelii, and other species of Plasmodium falciparum (Plasmodium vivax, Plasmodium berghei, Plasmodium falciparum), The acquisition of a large number of genomic sequences laid a solid foundation for further research on Plasmodium. The analysis of gene function and proteomics have become the main tasks in the post-genomic era. Through the discovery and identification of a large number of differential genes and proteins related to the invasion of plasmodium sporozoite liver cells, It will be helpful to understand the molecular mechanism of malaria parasite growth and development, to provide molecular theoretical basis for malaria control, to provide new targets for drugs and vaccines, and to lay a solid foundation for the adoption of control strategies to overcome malaria. So this experiment is based on the model of Anopheles stephens-Plasmodium yoelii. The gene and protein aspects of sporozoite invading hepatocyte related molecules were studied. On the one hand, PCR differential display RT-PCR (DD-PCR) technique was used to screen the differential cDNAbetween salivary gland and oocyst sporozoite of Plasmodium yoelii. After comparative analysis, the difference of cDNAs between salivary glands and oocysts of Plasmodium yoelii was obtained. Salivary gland sporozoite specific differential sequence, Cloning into TA vector, according to sequence homology analysis and functional prediction, we speculated that the gene may be related to the invasion of hepatocytes by sporozoite. On the other hand, two-dimensional gel electrophoresis was used.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R383

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