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.

目的 探讨DNA疫苗免疫接种方法对抗体水平及辅助性T细胞极化的影响。方法 利用DNA重组技术构建恶性疟原虫红内期多表位nNA疫苗，通过基因枪、肌肉注射、皮内注射等接种方法免疫小鼠，红ELISA测定该疫苗诱导产生特异性抗体的总量、亚型及表位特异性，比较不同免疫接种方法对DNA疫苗诱导产生抗体及辅助性T细胞极化的影响。结果 接种DNA疫苗的小鼠经3次免疫后都产生了较高滴度的抗重组蛋白抗体。基因枪组单位DNA诱导抗体滴度是肌注组的120倍。基因枪组小鼠血清抗体以IgG1升高为土，而肌注和皮内注射组小鼠血清抗体均以IgG2a升高为主。各免疫接种组诱导产生的抗表位多肽抗体特异性相同。结论 不同免疫接种方法不但会影响血清总免疫球蛋白水平，还能使辅助性T细胞向不同方向极化。肌注和皮内注射诱导产生以IgG2a为主的TH1型免疫应答，基因枪接种诱导产牛以IgGl为主的TH2型免疫应答。DNA疫苗的摄取方式可明显影响辅助性T细胞的极化。

Objective. To evaluate the Plasmodium falciparum CTL epitopc vaccines in HLA class I allele specific human cell lines that have high frequency among Chinese population. Methods. Synthesized oligonucleotides encoding for P. f. CTL epitope genes, constructed eukaryotic expression plasmids, transfcctod the minigenes into HLA class I allele specific human cell lines and identified endogenous expressing of the minigenes by RT-PCR and HLA stabilization aasay. Results. Two mini-genes encoding Plasmodium faleiparum CTL epitopes were designcd and cloned, respectiveIy, into an eukaryotic expressing vector to form TR26 which was restricted to HLA-B51, SH6 which was rcstricted to HLA-A2. 1, and TS, which had the two aforementioned mini-genes fused in tandem. All of these CTL epitope genes were transfected and endogenously expressed in respective cell lines containing appropriate HLA molecules. The obviously increased expressions of HLA class I molecules were detected in the transfected cell lines. It was demonstrated that the two discrete Plasmodium falciparum epitope genes were effectivcly processed and presented, and the close proximity of the two epitope genes in one chain as in mini-gene TS did not interfere with the processing and presenting of each epitope gene in corresponding cell line. Conclusion. A successful exprcssion and presentation of multiple CTL epitope mini-gene in MHC class I allele specific human ceU lines were demonstrated by an in vitro assay, which could be corresponding to the vaccination of CTL vaccines in people with different MHC I molecules. This work also suggested the possibility of constructing a muhiple CTL epitope plasmodium falcipa~m DNA vaccine that could cover most of Chinese population.

Objective Four B and Th cell epitopas were selected from conservative domain of Plasmodium falciparum antigens to construct two groups of chimeric malaria DNA vaccines with different configurations and their antigenicitles were studied. Methods The partially synthesized oligonucleotide was annealed, PCR amplified and cloned into a mammalian cell expression vector. By using a pair of isocaudamers on the vector, different single copies of B epitopes were multiplied and were tenderly stdnged into two groups of chimeric DNA vaccine with different configurations, BALB/c mice were immunized with these DNA plasmids by either intramuscular or intraderrnal injections. Results The antisera from the immunized mice tested by ELISA showed that only the configuration which had a single copy of universal T helper cell epitope, CS. T3, located at the C terminal of the multi-copy B ceil epitopes induced a high antibody response. The T helper cell epitope at any other position of the peptide, or the double T helper cell epitopes configured with the B cell epitopes did not enhance antibody response, and some configurations even decreased the hurnoral response to a B cell epitope. Conclusion This study demonstrated that both combination and configuration of the epitope may affect the antigenicity of a chimeric multiple antigen.

同尾酶是一类识别不同核菁酸序列但能酶切产生相同粘性末端的限制性内切酶，依靠同尾酶的这种特性，可以根据需要将不同的DNA片段进行灵活组合，获得各种排列顺序的多价表位重组疫苗。将这种方法用于疟疾多价重组DNA疫苗的研制；BALB/c小鼠免疫实验对所得重组疫苗PU286的免疫原性进行了测定。