Analyisis of the Plasmodial rhoptry protein function by gene targeting

通过基因打靶分析疟原虫棒状体蛋白功能

基本信息

批准号：
12670232
负责人：
TORII Motomi
金额：
$ 2.18万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670232/
关键词：
malaria Plasmodium merozoite invasion gene-targeting ジーンターゲティングロプトリー蛋白

项目摘要

Invasive stage of the malaria parasite contains characteristic microorganelles at the apical end. One of the organelle is called as the rhoptries and thought to be involved in the critical invasion step into the host cells. The aim of this research proposal was to analyze the function of rhoptry proteins (140-kDa RhopHl and 100-kDa RhopHS proteins) by using gene-targeting technique. Because the gene-targeting technique in rodentplasmodium was only established in P. berghei, closely related species to P. yoelii, we evaluated; 1) the sensitivity to the anti-plasmodial drug, pyrimethamine, for the P. yoelii clone we maintain, 2) the efficacy of the gene-introduction into P. yoelii by transfecting the circular form of pMD204, which carry the resistance to pyrimethamine, into P. yoelii, 3) the efficient concentration of pyrimethamine for pMD204-transfected P. yoelii. The results suggested that the transfection into P. yoelii could be achieved with a slightly modified technique from that into P. berghei. The first version of the gene-targeting constructs were designed for P yoelii rhoptry proteins, PyRhopHl and PyRhopHS, and constructed by inserting PCR-amplified DNA fragments into the targeting vector pMD204 (pYRH1-Dis and pYR3-Dis, respectively). The attempt to transfect the linearized genetargeting constructs into P. yoelii and obtain the gene-disrupted clones under drug pressure were unsuccessful at this moment. To overcome this problem, we re-designed the gene-targeting constructs and the attempts to disrupt these gene loci is still underway.

疟疾寄生虫的侵入性阶段在顶端含有特征性的微型制剂。其中一个细胞器被称为rhoptries，并被认为与临界入侵相关。这项研究建议的目的是通过使用基因靶向技术分析Rhoptry蛋白（140-KDA Rhophl和100 kDa Rhophs蛋白）的功能。由于啮齿动物质量流菌中的基因靶向技术仅在Berghei P. berghei中建立，因此与P. yoelii紧密相关的物种，我们进行了评估。 1）对我们维持的P. yoelii克隆的抗质子药物的敏感性，2）通过转染PMD204的圆形形式，将基因 - 引入基因 - 引入基因的疗效，该形式具有对吡胺胺，吡胺甲胺，耐药性，进入P. yoelii，3）PMD204转染的P. yoelii的嘧啶的有效浓度。结果表明，可以通过从该杆菌稍微修改的技术转染到P. yoelii中。基因靶向构建体的第一个版本是为P Yoelii Rhoptry蛋白，pyRHophl和Pyrhophs设计的，并通过将PCR放大的DNA DNA片段插入靶向载体PMD204（分别分别为Pyrh1-dis和Pyr3-Dis）来构建。目前，在药物压力下，将线性化基因射击构建体转染并获得基因脱离的克隆的尝试尚未成功。为了克服这个问题，我们重新设计了靶向基因的构建体，并仍在进行破坏这些基因基因座的尝试。