High-throughput screening of novel malaria vaccine candidates using human immune sera

使用人类免疫血清高通量筛选新型疟疾候选疫苗

• 批准号: 16406009
• 负责人: TSUBOI Takafumi
• 金额: $ 8.45万
• 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: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16406009/
• 关键词: malaria; vaccine; genome; biotechnology; protein; serum; 感染症; 国際研究者交流; タイ; 無細胞タンパク質合成

Malaria remains one of the leading causes of both morbidity and mortality in humans residing in the tropical countries. The evidence of increasing resistance of the Plasmodium falciparum parasite to chemotherapeutic agents highlights the critical need for an effective vaccine. After the establishment of malaria genome database, we have now free access to the genome data to search for novel vaccine candidates. However, one of the bottlenecks for the malaria vaccine research is the difficulty of the recombinant protein expression using conventional methods. We applied a high-throughput protein production method based on the wheat germ cell-free system to the malaria vaccine research. To identify novel malaria vaccine candidates, we selected and cloned more than 180 genes which are expected to be expressed in merozoite stage, then prepared transcription templates through PCR-based high throughput procedures, followed by protein synthesis by wheat germ cell-free system. Using this approach, we succeeded in obtaining 149 recombinant merozoite proteins. At the same time, we successfully obtained approximately 30 human immune sera from asymptomatic villagers living along the Thai-Myanmar border, and approximately 30 symptomatic malaria patient sera from hospital in Bangkok, Thailand. All human serum samples used in this study were reviewed and approved by the Institutional Ethics Committee of the Thai Ministry of Public Health and of the Ehime University, Japan. Finally, we tried to screen these recombinant proteins for identifying novel malaria vaccine candidates with five cases of the malaria immune sera by ELISA. As a result of this preliminary screening, we identified ten merozoite proteins as antigens. Accordingly, this strategy using wheat germ cell-free system will be a major breakthrough in the novel malaria vaccine candidate discovery research.

疟疾仍然是居住在热带国家的人类发病率和死亡率的主要原因之一。恶性疟原虫寄生虫对化学治疗剂的耐药性增加的证据突显了对有效疫苗的迫切需求。建立疟疾基因组数据库后，我们现在可以免费访问基因组数据，以搜索新型疫苗候选物。但是，疟疾疫苗研究的瓶颈之一是使用常规方法的重组蛋白表达的困难。我们将基于小麦生殖细胞系统的高通量蛋白生产方法应用于疟疾疫苗研究。为了鉴定新的疟疾疫苗候选物，我们选择并克隆了180多个基因，预计将以梅洛唑群体阶段表达，然后通过基于PCR的高通量程序制备的转录模板，然后通过无小麦生殖细胞系统合成蛋白质。使用这种方法，我们成功获得了149种重组蛋白蛋白。同时，我们从居住在泰国山脉边界的无症状村民中成功获得了大约30种人类免疫血清，并从泰国曼谷的医院获得了大约30个有症状的疟疾患者血清。这项研究中使用的所有人类血清样本均由泰国公共卫生部和日本埃希姆大学的机构伦理委员会审查和批准。最后，我们试图筛选这些重组蛋白，以鉴定ELISA的五例疟疾免疫血清的新型疟疾疫苗候选物。由于这种初步筛选，我们确定了十种蛋白蛋白是抗原。因此，这种使用无小麦生殖细胞系统的策略将是新型疟疾疫苗候选发现研究的主要突破。