Analyisis of the Plasmodial rhoptry protein function by gene targeting

疟疾寄生虫的侵袭期在顶端含有特有的微细胞器。其中一个细胞器被称为棒状细胞器，被认为参与了进入宿主细胞的关键入侵步骤。本研究的目的是利用基因打靶技术分析杆状病毒蛋白(140 kDa Rhop H1和100 kDa Rhop HS蛋白)的功能。由于基因打靶技术仅在与约氏疟原虫关系密切的伯氏疟原虫中建立，因此我们评估了：1)我们维持的约氏疟原虫克隆对抗疟原虫药物乙胺胺的敏感性，2)通过将携带乙胺胺抗性的环状pMD204基因导入约氏疟原虫的有效性，3)对pMD204转基因的约氏疟原虫的有效浓度。结果表明，用一种稍加改进的方法即可将其转入约氏疟原虫。第一种基因打靶载体是针对约氏杆菌杆状病毒蛋白，即pYRHopH1和pYRHopHS设计的，并通过将PCR扩增的DNA片段插入到打靶载体pMD204(分别为pYRH1-Dis和pYR3-Dis)中而构建。将线性化的基因靶向构建体导入约氏青霉菌，并在药物压力下获得基因中断克隆的尝试目前尚未成功。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.