SERINE PROTEASES AND IMMUNITY IN ANOPHELES GAMBIAE

冈比亚按蚊的丝氨酸蛋白酶和免疫

• 批准号: 6632038
• 负责人: SUSAN M PASKEWITZ
• 金额: $ 28.8万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6632038
• 关键词: Anopheles; SDS polyacrylamide gel electrophoresis; arthropod borne communicable disease; bioassay; communicable disease control; communicable disease transmission; cytogenetics; disease vectors; enzyme activity; enzyme induction /repression; gene expression; genetic mapping; genetic transcription; host organism interaction; humoral immunity; in situ hybridization; ion exchange chromatography; laboratory mouse; laboratory rabbit; messenger RNA; molecular cloning; northern blottings; polymerase chain reaction; quantitative trait loci; serine proteinases; southern blotting

Malaria, a disease caused by parasitic protozoans in the genus Plasmodium, is transmitted by mosquitoes to humans. Each year, hundreds of millions of people are infected with this organism and 2-3 million fatalities result, mostly in young children. The problem is most severe in Africa, and is now worsening because of the increasing resistance of parasites to the inexpensive drugs used for prevention and control in the past. A second important strategy for control, reducing mosquito populations through insecticide use, is also threatened because of insecticide resistance, increasing costs and loss of trained personnel. A new target for research has been the interaction between the mosquito and the parasite. The long-term goal of the research presented here is to understand the molecular basis for incompatibility between mosquitoes and malaria parasites because this may suggest ways to enhance mechanisms that cause incompatibility through genetic or chemical manipulation of vector mosquitoes. The proposed research will focus on characterizing proteins and genes that are involved in the mosquito's humoral immune responses, which can lead to the death of parasites. In particular, serine proteases are important enzymes in generation of two parasite-killing mechanisms, melanotic encapsulation and the antimicrobial response. In the African malaria vector, Anopheles gambiae, three serine protease genes, AgSp14D1, AgSp14D2, and AgSp14A, show changes in transcript levels after bacterial injections or infection with malaria parasites. These molecules will be characterized and tested for involvement in immune responses against parasites in vitro and in vivo. In addition, prophenoloxidase activating serine proteases, which are important in triggering parasite melanization, will be biochemically purified and then cloned and characterized. Finally, several A. gambiae serine proteases that exhibit domain structures similar to other enzymes involved in immune responses will be characterized at the mRNA and protein levels.

疟疾是一种由疟原虫属寄生原生动物引起的疾病，通过蚊子传播给人类。每年，数亿人感染这种微生物，导致 2-300 万人死亡，其中大多数是幼儿。这一问题在非洲最为严重，而且由于寄生虫对过去用于预防和控制的廉价药物的抵抗力不断增强，该问题现在正在恶化。第二个重要的控制策略，即通过使用杀虫剂减少蚊子数量，也由于杀虫剂耐药性、成本增加和训练有素的人员流失而受到威胁。研究的新目标是蚊子和寄生虫之间的相互作用。这里提出的研究的长期目标是了解蚊子和疟疾寄生虫之间不相容性的分子基础，因为这可能提出通过对媒介蚊子进行遗传或化学操作来增强导致不相容性的机制的方法。拟议的研究将重点关注参与蚊子体液免疫反应的蛋白质和基因的特征，这可能导致寄生虫死亡。特别是，丝氨酸蛋白酶是产生两种寄生虫杀灭机制（黑素包囊和抗菌反应）的重要酶。在非洲疟疾载体冈比亚按蚊中，三种丝氨酸蛋白酶基因 AgSp14D1、AgSp14D2 和 AgSp14A 在细菌注射或感染疟原虫后表现出转录水平的变化。这些分子将被表征和测试是否参与体内和体外针对寄生虫的免疫反应。此外，激活丝氨酸蛋白酶的原酚氧化酶对引发寄生虫黑色化很重要，将进行生化纯化，然后进行克隆和表征。最后，几种冈比亚阿丝氨酸蛋白酶的结构域结构与参与免疫反应的其他酶相似，将在 mRNA 和蛋白质水平上进行表征。