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蛋白，在基于这些蛋白的疫苗开发之前，必须评估这些蛋白在现场分离株中的表达情况。本项目的目的是分析参与红细胞侵袭过程的多基因家族EBL、RBL和Clag/Rhop H1编码的红细胞结合蛋白的mRNA和蛋白表达。我们在泰国采集了恶性疟原虫的野外分离株，并对成熟裂殖体进行了短期培养和浓缩。利用针对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进行初步分析，我们发现这些基因家族成员是高度多样化的，需要精心设计寡核苷酸来区分每个成员，但识别个别成员。我们生成了一组针对每个成员的抗血清，并分析了每个成员的表达水平。对于Clag/Rhop H1，成员间呈现多样性的区域在不同分离株之间也表现出多态，这表明需要产生区分每个成员的抗血清，但对所有类型的等位基因都有反应。

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.