Genome-wide screening of the novel malaria transmission-blocking vaccine candidates

疟疾仍然是居住在热带国家的人类发病和死亡的主要原因之一。有证据表明，恶性疟原虫对氯喹等化疗药物的抗药性不断增强，这突显了对有效疫苗的迫切需要。然而，迄今为止，有效的疟疾疫苗从未实现。自从恶性疟原虫基因组序列和特定阶段蛋白质组数据于2002年10月完成以来，我们现在可以自由访问基因组数据来搜索新的候选疫苗。然而，疫苗开发的瓶颈之一是恶性疟原虫外显子中AT含量较高，这将大大抑制传统方法下重组蛋白的表达。我们的策略是根据恶性疟原虫基因组序列表达每种蛋白的重组蛋白，而不使用人工合成的基因。传播阻断疫苗(TBV)防止…传播通过诱导针对在有性期寄生虫上特异表达的抗原的抗体，更多地感染疟疾。由于具有良好特性的TBV候选病毒只有四个(Pfs25、Pfs28、Pfs48/45和Pfs230)，为了成功地开发出TBV，有必要准备尽可能多的新的TBV候选者。为了识别新的TBV候选基因，我们从基因组和转录组数据库中搜索了192个基因，这些基因预计只在恶性疟原虫配子体阶段表达。将这些基因克隆到质粒中，通过基于PCR的程序制备模板进行转录，然后在小麦胚芽无细胞系统中高通量合成重组蛋白。利用这种方法，我们成功地获得了120个重组蛋白。在用抗配子体单抗筛选这些重组蛋白以鉴定新的TBV候选蛋白后，我们鉴定了一个表达在配子期表面的新抗原。较少

Malaria remains one of the leading causes of both morbidity and mortality of humans residing in the tropical countries. The evidence of increasing resistance of the Plasmodium falciparum parasite to chemotherapeutic agents, such as chloroquine, highlights the critical need for an effective vaccine. However, the effective malaria vaccine has never been achieved to date. Since the genomic sequence of P.falciparum together with stage-specific proteome data was completed in October 2002, we have now free access to the genome data to search for novel vaccine candidates. However, one of the bottlenecks for the vaccine development is the high AT content in exons of P.falciparum, which will considerably inhibit the recombinant protein expression using conventional methods. Our strategy is to express recombinant proteins for the characterization of each protein based on the genome sequences of P.falciparum without using synthetic gene. Transmission-blocking vaccines (TBVs) prevent the transmiss … More ion of malaria by inducing antibodies against antigens specifically expressed on the sexual stage parasites. Since well-characterized TBV candidates are only four (Pfs25, Pfs28, Pfs48/45, and Pfs230), it would be necessary to prepare novel TBV candidates as many as possible for a successful TBV development. In order to identify the novel TBV candidates, we searched a combined dataset from genome and transcriptome databases and we selected 192 genes, which are expected to be expressed only in gametocyte stage of P.falciparum. These genes were cloned into plasmids and templates 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 120 recombinant proteins. After the screening of these recombinant proteins to identify novel TBV candidates with anti-gametocyte monoclonal antibodies, we have identified a novel antigen expressed on the surface of gametocyte stage. Less