Comprehensive screening and identification of the novel malaria transmission-blocking vaccine candidate antigens

新型疟疾传播阻断疫苗候选抗原的综合筛选和鉴定

基本信息

批准号：
18390129
负责人：
TSUBOI Takafumi
金额：
$ 10.49万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18390129/
关键词：
malaria vaccine infectious diseases biotechnology genome International scientist exchange Thailand

项目摘要

Malaria transmission-blocking vaccines (TBVs) prevent the transmission of malaria by inducing antibodies against antigens specifically expressed on the sexual stage parasites. Since well-characterized TBV candidates are only four, it would be necessary to identify novel TBV candidates for a successful TBV development. In order to identify the novel TBV candidates, we searched a combined dataset from genome and transcriptome databases and selected 192 genes, which are expected to be expressed only in gametocyte stage of P. falciparum. These genes were cloned into plasmids and 170 of the template cDNA clones were prepared for transcription through PCR-based procedures, followed by high throughput recombinant protein synthesis by wheat germ cell-free system. Using this approach, we succeeded in obtaining 148 recombinant proteins. After the screening of these recombinant proteins to identify novel TBV candidates with malaria patient sera harboring parasite transmission-blocking antibodies, we have identified 27 novel antigens. After subclone these candidate genes into pEU plasmid for the large-scale expression using the wheat germ cell-free system, we have expressed and affinity purified 22 targets. In order to obtain antisera, we immunized these proteins into mice. After the confirmation of the immunoreactivity of these antisera against parasites, we examined the transmission-blocking efficacy of the sera. Finally we identified the two novel parasite antigens, which induced transmission-blocking antibodies in mice. Accordingly, this approach will be useful for the novel transmission-blocking vaccine candidate discovery.

疟疾传播阻断疫苗（TBVS）通过诱导针对在性阶段寄生虫上明确表达的抗原的抗体来防止疟疾传播。由于特征良好的TBV候选者只有四个，因此有必要确定成功的TBV开发的新型TBV候选者。为了识别新型TBV候选者，我们搜索了一个来自基因组和转录组数据库的组合数据集以及选定的192个基因，预计仅在恶性疟原虫的配子细胞阶段表达。将这些基因克隆到质粒中，并通过基于PCR的程序制备170个模板cDNA克隆，然后通过无小麦生殖细胞系统进行高吞吐量重组蛋白合成。使用这种方法，我们成功获得了148种重组蛋白。在筛选了这些重组蛋白以鉴定出具有疟疾患者血清具有寄生虫传播抗体抗体的新型TBV候选者后，我们已经鉴定出27种新型抗原。在亚克隆中，这些候选基因使用无小麦生殖细胞系统进行大规模表达中的PEU质粒后，我们表达并亲和力纯化了22个目标。为了获得抗血清，我们将这些蛋白质免疫到小鼠中。在确认这些抗血清对寄生虫的免疫反应性后，我们检查了血清的传输阻滞功效。最后，我们确定了两种新型的寄生虫抗原，它们诱导了小鼠的透射阻滞抗体。因此，这种方法对于新型的传输阻滞疫苗候选发现将很有用。