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Production of human monoclonal antibodies, which inhibit in vitro growth of Plasmodium falciparum

生产抑制恶性疟原虫体外生长的人单克隆抗体

基本信息

批准号：
18590407
负责人：
TACHIBANA Hiroshi
金额：
$ 2.46万
依托单位：
Tokai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
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-18590407/
关键词：
malaria Plasmodium falciparum human monoclonal antibod Fab merozoite surface protein 1

项目摘要

An effective vaccine for malaria has not yet been developed. Passive immunotherapy with human monoclonal antibodies may provide a valuable therapeutic alternative. A combinatorial immunoglobulin gene library was constructed from lymphocytes of patients infected with Plasmodium falciparum and screened for the production of human monoclonal antibodies to the C terminal 19-kDa fragment of P. falciparum merozoite surface protein 1 (MSP-1_<19>). MSP-lis-specific Fab fragments were produced in bacterial expression system. Immunofluorescence staining of P. falciparum merozoites by the Fab fragments was demonstrated on FCR3 and 3D7 strains, which were representatives of dimorphic allelic variants in MSP-1_<19>, suggesting the Fab's reativity to a conserved region. To examine whether the epitope for these Fabs was recognized by immune sera, competition ELISA was also performed using sera from ten malaria-immune individuals in the Solomon Islands or plasmas from eight donors of lymphocytes. Three of the immune sera and three of donors' plasmas showed significant inhibition compared with control sera. The effect of single amino acid modifications in the third complementarity-determining regions of the heavy and light chains on affinity was examined in one of the Fab fragments, P125. Recombination PCR was used to modify Tyr^<92> or Ile^<97> in the light chain and Val^<101> or Trp^<107> in the heavy chain. No effective replacements for Tyr^<92> and Val^<101> were found, but possible substitutions of 11e^<97> with Gly, Leu, Glu, Ala and Ser, and of Trp^<107>with Arg and Ser were demonstrated. Of these modified Fab fragments, the affinities of Fabs with Ile^<97>Leu and Typ^<107>Ser mutations were slightly higher than that of the original Fab. The modified antibodies may be applicable to analyze epitope structures of MSP-1_<19>.

目前还没有研制出有效的疟疾疫苗。被动免疫治疗与人类单克隆抗体可能提供一个有价值的治疗选择。从恶性疟原虫感染患者的淋巴细胞中构建了一个组合免疫球蛋白基因文库，并筛选了抗恶性疟原虫裂殖子表面蛋白1（MSP-1）C端19 kDa片段的人源单克隆抗体<19>。在细菌表达系统中产生MSP-lis特异性Fab片段。在MSP-1_1的二型等位变异体FCR 3和3D 7株上证实了Fab片段对恶性疟原虫裂殖子的免疫荧光染色<19>，表明Fab片段与保守区域反应。为了检查这些Fab的表位是否被免疫血清识别，还使用来自所罗门群岛的10个疟疾免疫个体的血清或来自8个淋巴细胞供体的血浆进行竞争ELISA。与对照血清相比，三种免疫血清和三种供体血浆显示出显著的抑制。在Fab片段之一P125中检查重链和轻链的第三互补决定区中的单个氨基酸修饰对亲和力的影响。使用扩增PCR修饰轻链中的Tyr^<92>或Ile^<97>和重链中的瓦尔^<101>或Trp^<107>。没有发现Tyr^和瓦尔^的有效替代<92><101>，但证明了11 e ^可能<97>被Gly、Leu、Glu、Ala和Ser取代，Trp^<107>可能被Arg和Ser取代。在这些修饰的Fab片段中，具有Ile^<97>Leu和Typ^<107>Ser突变的Fab的亲和力略高于原始Fab的亲和力。修饰后的抗体可用于MSP-1的表位结构分析<19>。