Analysis of the protein expression level of the erythrocyte binding proteins in Plasmodium falciparum field isolates.

红细胞结合蛋白是有效的疟疾疫苗候选物，并且在基于这些蛋白的疫苗开发之前必须评估这些蛋白在田间分离株中的表达。本项目的目的是分析红细胞结合蛋白的mRNA和蛋白质表达，由多基因家族EBL，RBL和Clag/RhopH 1编码，它们参与红细胞侵袭过程。我们在泰国收集了恶性疟原虫田间分离株，并进行了短期培养和富集成熟菌株。用靶向每个多基因家族成员的寡核苷酸进行RT-PCR检测转录水平。然而，通过对这些基因的多态性分析，我们发现这些基因家族成员是高度多样化的，需要仔细设计寡核苷酸来区分每个成员，但要识别单个成员。我们针对每个成员产生了一组抗血清，并分析了每个成员的表达水平。Clag/RhopH 1在成员间表现出多样性的区域在分离株间也表现出多态性，这表明需要产生区分每个成员但反应所有类型等位基因的抗血清。

Erythrocyte binding proteins are potent malaria vaccine candidates and the expression of these proteins in the field isolates has to be evaluated before vaccine development based upon these proteins. The objective of this project is to analyze mRNA and protein expression for the erythrocyte binding proteins, encoded by multigene family EBL, RBL, and Clag/RhopH1, which are involved in the erythrocyte invasion process. We collected P.falciparum field isolates in Thailand and cultured for short period and enriched mature schizonts. Using oligonucleotide targeting members of each multigene family, RT-PCR was performed to detect transcripts level. However, by preliminal analysis of the polymorphism of these genes, we found these gene family members are highly diversified and carefull design of the oligonucleotide is required to distinguish each member, but identify individual members. We generated a panel of antisera against each members and analyzed the expression levels of the each member. For Clag/RhopH1, the region showing diversified each other among members shows also polymorphic among isolates, indicated that it is required to generate antisera which distinguish each members, but react all type of the allele.