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Malarial development in mosquito and vaccine development. Mechanism of malarial sporozoite accumulation in the salivary glands of Anopheline mosquitoes.

蚊子中的疟疾发展和疫苗开发。

基本信息

批准号：
06045019
负责人：
ANDO Katsuhiko
金额：
$ 3.9万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

We studied the mechanism of malarial sporozoite accumulation of Plasmodium berghei in the salivery gland of Anopheles stephensi.1. We got following results with the observation of midgut infected with Plasmodium berghei by transmission electron microscope (TEM).(1) Ookinate reached the basal lamina of midgut through the cell junction 24 hours after blood was ingested. (2) Two days after infection, ookinate whose diameter was 6 um has stopped moving and became round. (3) Eight days after infection, there were many nuclei which developed into sporozoites inside the oocyst. (4) Fourteen days after infection, oocyst developed matured oocyst, and many sparozoite exsisted around the core.2. To observe the penetration of the sporozoites into salivary glands, we fixed living mosquitoes by 2.5% osmium and dissected them to remove the salivary glands after 2 hours. We got following results by scanning electron microscope and TEM.(1) Sporozoites exsisted on the surface and some of them were penetrating in the salivary glands. (2) Inside of the salivary glands, sporozoites entered the cytoplasm of the cell from outside and gathered in the secretory cavity.3. We extracted mRNA from salivery gland of mosquito and synthesized cDNA.The cDNA was ligated lambdagtll phage and cDNA libraries were produced. These were screened by the monoclonal antibody of salivery gland and 4 possitive clones were selected. The size of insert of 4 clones were 1450bp, 360bp, 220bp and 790bp.

本实验研究了伯氏疟原虫子孢子在斯氏按蚊唾液腺中聚集的机制。用透射电镜观察了伯氏疟原虫感染后的中肠，结果如下：(1)Ookinate在血液摄入后24小时通过细胞连接到达中肠的基底层。(2)感染后第2天，卵细胞停止运动，变圆，直径约6 μ m。(3)感染后第8天，卵囊内出现大量的核，并发育成子孢子。(4)感染后14d，卵囊发育成熟，核心周围有大量的孢子.为了观察子孢子对唾液腺的穿透，我们用2.5%锇固定活蚊，并在2小时后解剖它们以去除唾液腺。通过扫描电子显微镜和透射电子显微镜观察，得到了以下结果。(1)唾液腺表面有子孢子，部分子孢子可穿透唾液腺。(2)在唾液腺内，子孢子从细胞外进入细胞质，聚集在分泌腔内.从蚊虫唾液腺中提取mRNA，合成cDNA，连接噬菌体，构建cDNA文库。经唾液腺单克隆抗体筛选，得到4个阳性克隆。4个克隆的插入片段大小分别为1450bp、360bp、220bp和790bp。