感染约氏疟原虫大劣按蚊消减文库的构建和差异表达基因的鉴定及分析

发布时间：2018-04-23 10:02

本文选题：抑制消减杂交 + 大劣按蚊　；参考：《第三军医大学》2005年硕士论文

【摘要】：疟疾是世界上主要的传染病之一,在全世界人群中具有很高的发病率和致病率。全球受疟疾威胁的人口超过30 亿,每年死亡的人数超过100 万(WHO:World Malaria Report 2005)。随着疟原虫多药抗性株的出现与迅速扩散、蚊媒对杀虫剂抗性的增加,以及目前尚无有效的抗疟疫苗,因此疟疾的控制工作将面临巨大的挑战,迫切需要发展新的疟疾控制策略。20 世纪90 年代初,WHO 提出了“遗传改造蚊媒”的疟疾防治新策略,而寻找按蚊抗疟原虫感染相关基因,克隆、鉴定并分析其功能,是实现这种新策略的首要工作。按蚊的先天免疫反应主要通过黑化包被反应的激活, NO 和抗菌肽的产生等机制抑制体内疟原虫的发育。但是NO 和抗菌肽只能在一定程度上调节按蚊体内的疟原虫感染率,而黑化包被反应则能完全抑制疟原虫的感染,是能否阻断按蚊感染疟原虫的最主要免疫机制。当疟原虫表面分子与按蚊的可溶性模式识别受体结合而触发该反应,并激活丝氨酸蛋白酶(Serine proteinase,Sp)级联,最终导致前酚氧化酶(Prophenoloxidase,PPO)分解激活酚氧化酶(Phenoloxidase,PO),PO 激活后续成分介导蛋白质发生交联并聚合成黑色素从而固定、隔离或杀死入侵的病原体。目前,已从冈比亚按蚊(Anopheles gambiae)克隆到几种不同的PPO、Sp 和抗菌肽等。但是究竟如何启动了按蚊抗疟原虫感染的免疫反应,目前仍不清楚。大劣按蚊是我国和东南亚地区的重要传疟蚊媒。研究发现,对恶性疟原虫敏感的大劣按蚊可以炓化包被约氏疟原虫卵囊,所以大劣按蚊-约氏疟原虫模型是研究按蚊抗疟原虫感染机制较好的模型。目前研究按蚊抗疟原虫感染相关分子采用的技术有退化PCR、DD-PCR 等,通过这些方法获得基因的数目有限,而且不能克服基因上调。本实验以大劣按蚊-约氏疟原虫为模型,利用新近建立的SSH 方法构建感染约氏疟原虫大劣按蚊消减文库,通过对差异基因的鉴定和生物信息学分析,预测其在按蚊感染疟原虫的过程中所起的作用,并对与先天免疫相关的差异基因进行半定量实验,分析约氏疟原虫感染前后其转录的变化,探讨它与按蚊抗疟原虫感染的先天免疫反应的关系。实验内容和结果主要包括以下方面:
[Abstract]:Malaria is one of the major infectious diseases in the world. More than 3 billion people worldwide are threatened by malaria, and more than 1 million people die each year at World Malaria Report 2005. With the emergence and rapid spread of multidrug resistant strains of Plasmodium, the increased resistance of mosquito vectors to insecticides, and the absence of effective antimalarial vaccines, malaria control will face enormous challenges. There is an urgent need to develop a new malaria control strategy. In the early 1990s, WHO put forward a new strategy for malaria control with "genetic transformation of mosquito vectors", and searched for genes related to the resistance of Anopheles to Plasmodium infection, cloned, identified and analyzed its function. Is the most important task to realize this new strategy. The innate immune response of Anopheles japonicus is mainly through the activation of melanized envelope reaction, the production of no and antimicrobial peptides, and so on, which inhibits the development of Plasmodium in vivo. However, no and antimicrobial peptides can only regulate the infection rate of malaria parasite in Anopheles mosquitoes to a certain extent, while the melanized coating reaction can completely inhibit the infection of malaria parasites, which is the main immune mechanism to block the infection of Anopheles mosquitoes. When the plasmodium surface molecules bind to the Anopheles soluble pattern recognition receptor, the reaction is triggered and the serine proteinase spa cascade is activated. In the end, Prophenoloxidase (PPO) decomposition activates the subsequent components of Phenoloxidase (PPOP) and mediates the cross-linking of proteins and polymerization into melanin to fix, isolate or kill the invading pathogens. At present, several different PPOSp and antimicrobial peptides have been cloned from Anopheles gambiaeae, Anopheles gambiaeae. But how the Anopheles infestation immune response against Plasmodium infection remains unclear. Anopheles dirus is an important mosquito vector in China and Southeast Asia. It was found that the Anopheles inferioris sensitive to Plasmodium falciparum could be encapsulated with Plasmodium yoelii oocysts, so the Anophele-Plasmodium malarial model was a better model to study the mechanism of Anopheles infestation against Plasmodium falciparum infection. At present, the techniques used to study Anopheles repens against Plasmodium infection include degenerated PCRN DD-PCR. The number of genes obtained by these methods is limited, and the gene upregulation cannot be overcome. Using Anophele-Plasmodium yoelii as a model, a subtractive library of Anopheles dirus infected with Plasmodium yoelii was constructed by using the newly established SSH method. The differential genes were identified and bioinformatics were analyzed. To predict the role of Anopheles in the infection of Plasmodium yoelii, and to analyze the transcription changes of Plasmodium yoelii before and after infection by semi-quantitative experiments of differentially related genes related to innate immunity. To investigate the relationship between Anopheles and the innate immune response of Anopheles to Plasmodium infection. The contents and results of the experiment mainly include the following aspects:
【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

【共引文献】