A novel dynamin-like GTPase gene, Pfdyn1, was cloned from an asexual stage cDNA library of Plasmodium falciparum Dd2 strain. Pfdyn1 contains a highly conserved N-terminal tripartite GTPase domain, a coiled-coil region, and a C-terminal 129 aa unknown function domain. Like yeast Vps1p, it lacks pleckstrin homology domain and proline-rich region. Western blot analysis showed that Pfdyn1 is a Triton X-100 insoluble protein expressed only in the mature sub-stage. Morphological studies ndicated that Pfdyn1 is partly co-localized with PfGRP, a known ER-resident protein, and localizes diffusely with several membrane structures and a 60-100nm vesicle both inside and on surface of the parasites and also in the cytoplasm of infected erythrocytes. The dsRNA originated by C-terminus fragment of Pfdyn1 inhibits markedly the growth of P. falciparum parasite at the erythrocyte stage. Those data showed that Pfdyn1 is a conservative, membrane related protein and plays an essential role for the survival of Plasmodium parasite.

Developing a polyepitope vaccine which contains diverse antigenic types is a promising strategy to cope with the problem of malaria variation and diversity. However, arranging the peptides to produce the most effective immunogenicity remains a hurdle. In an attempt to develop an effective complex antigenic gene vaccine, we constructed a polyepitope library by randomly assembling epitopes using the epitope shuffling technique. The polyepitope library, which contains epitopes from different antigens of Plasmodium falciparum,was divided into five sub-libraries based on the size of chimeric genes. Here we report that higher antibody titers were observed in mice with immunized with sub-libraries containing genes >1200 bp, using enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IFAT) assay to determine both individual epitope peptides and the natural parasites at the erythrocyte stage. Different levels of IgG subtypes and cytokines were elicited by different sub-library and administration times. In a rodent malaria model, some groups of immunized mice were partially cross-protected against a lethal challenge from Plasmodium yoelii. These results suggest that the immunogenicity of a polyepitope chimeric antigen is ssentially conformation- and length-dependent, and demonstrates that the promising advantage of epitope shuffling technology is that it allows us to randomly assemble many polyepitope molecules in tandem format. This finding also indicates that polyepitope library vaccination is a suitable approach for screening optimized chimeric gene vaccines against malaria and other diseases.

目的 分离、克隆恶性疟原虫带7蛋白家族蛋白编码基因pfstom，并对其功能进行初步研究。方法 根据国际恶性疟研究公共数据库释放的已完成序列数据（CoDing Sequence，CDS数据）设计引物，用RT-PCR方法从恶性疟原虫中国海南株（FCCl/HN）中得到其带7蛋白家族蛋白基因cDNA--pfstom。应用多种软件对其结构功能、基因进化特征以及同源性分析。用pET30a系统表达其具有stomatin样蛋白结构域的C端蛋白，免疫新西兰大白兔并纯化抗体。Westem杂交检测其在FCCl/HN表达情况。结果 pfstom基囚编码区全长1125bp，编码374个氨基酸，C端具有和人红细胞整合膜蛋白带7蛋白家族中stomatin样蛋白类似的结构。进化特征分析显示：在带7蛋白家族中，Pfstom蛋白与stomatin样蛋白亲缘关系较近。Western杂交发现Pfstom蛋白在FCC1/HN的滋养体期呈期特异性表达，在与膜相关和与膜不相关的感染红细胞蛋白提取物中都存在。结论 Pfstom是带7蛋白家族蛋白，在FCC1/HN红内期的滋养体期呈特异性表达，环状体期不表达，该蛋白是膜相关蛋白。

Plasmodium falciparum CTL epitope minigenes containing HLA-A2 and HLA-B7 subtype supermotifs were cooned into a plasmid expression vector, this expression was measured in eight buman HLA class I molecule specific cell lines. Three assays for in vitro antigen prsentation analysis were developed to examine the cross-binding between CTL epitopesand HLA classI molecules, including cell surface peptide-MHC class I binding assay, bindingstabilization assay and MHC class I assembling assay. The results demonstrated that the HLA-B51 restricted CTL epitope of Plasmodium faiciparum could be presented by other HLA class I molecules; however, no other presentation was found for HLA-A2.1 CTL epitope. This work suggests the possibility for improved vaccine-coverage rates by development of a CTL vaccine which contains epitopes capable of cross-binding among different MHC class I alleles.