Analyisis of the Plasmodial rhoptry protein function by gene targeting

疟原虫的侵入阶段在顶端含有特征性的微细胞器。其中一种细胞器被称为棒状体，并被认为参与了入侵宿主细胞的关键步骤。本研究的目的是利用基因打靶技术分析棒状体蛋白（140-kDa RhopHl和100-kDa RhopHS蛋白）的功能。由于啮齿类疟原虫基因打靶技术仅在伯氏疟原虫中建立，而伯氏疟原虫与约氏疟原虫关系密切，因此我们进行了评价; 1）我们保持的约氏疟原虫克隆对抗疟原虫药物乙胺嘧啶的敏感性，2）通过将携带乙胺嘧啶抗性的pMD 204的环状形式导入约氏疟原虫而将基因导入约氏疟原虫的功效，3）乙胺嘧啶对pMD 204转染的约氏疟原虫的有效浓度。结果表明，对约氏疟原虫的转染技术稍加改进即可实现约氏疟原虫的转染。基因靶向构建体的第一个版本是针对约氏疟原虫棒状体蛋白PyRhopHl和PyRhopHS设计的，并且通过将PCR扩增的DNA片段插入靶向载体pMD 204（分别为pYRH 1-Dis和pYR 3-Dis）中来构建。将线性化的基因靶向构建体转染到约氏疟原虫中并在药物压力下获得基因破坏的克隆的尝试此时不成功。为了克服这个问题，我们重新设计了基因靶向结构，破坏这些基因位点的尝试仍在进行中。

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.