Production of high-affinity human monoclonal antibody fab fragments to the 19-kilodalton C-terminal merozoite surface protein 1 of Plasmodium falciparum
Production of high-affinity human monoclonal antibody fab fragments to the 19-kilodalton C-terminal merozoite surface protein 1 of Plasmodium falciparum
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DOI：
10.1128/iai.00062-07
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发表时间：
2007-07-01
影响因子：
3.1
通讯作者：
Tachibana, Hiroshi
中科院分区：
文献类型：
--
作者：
Cheng, Xun-Jia;Hayasaka, Hitoshi;Tachibana, Hiroshi
利用8例恶性疟原虫感染患者外周血淋巴细胞构建组合免疫球蛋白基因库，筛选制备恶性疟原虫merozoite surface protein 1(MSP-1) c端19 kda片段的人单克隆抗体Fab片段（19）。三个Fab克隆在非还原条件下识别重组MSP-1（19）。间接免疫荧光显微镜显示，三个Fab克隆染色了FCR3和3D7菌株的后期滋养体/分裂体和分裂体表面，表明Fab对保守的表位具有反应性。重链基因序列分析显示，与种系V段最接近的是VH1-8和VH7-81，同源性为91% ~ 98%。最接近的种系D段为D3-10，最接近的种系J段为JH4或JH5，同源性为90% ~ 97%。在轻链基因中，J kappa 2、J kappa 4和J kappa 5片段最接近种系V片段为A27。这些Fab片段对重组MSP-1（19）的解离常数在1.09 × 10(-9) ~ 2.66 × 10(-9) m之间。抗MSP-1（19）小鼠单克隆抗体12.8竞争性地抑制了这三个Fab片段与MSP-I-19的结合，抑制了分裂子对红细胞的侵袭。然而，亲和力最高的人Fab片段并没有抑制恶性疟原虫的体外生长。这是首次报道人恶性疟原虫MSP-1单克隆抗体的基因分析和细菌表达（19）。来自疟疾患者的组合免疫球蛋白基因库为生产恶性疟原虫特异性高亲和力人抗体提供了一个潜在的工具。
A combinatorial immunoglobulin gene library was constructed from peripheral blood lymphocytes of eight patients infected with Plasmodium falciparum and was screened for the production of human monoclonal antibody Fab fragments to the C-terminal 19-kDa fragment of P. falciparum merozoite surface protein 1 (MSP-1(19)). Three Fab clones recognized recombinant MSP-1(19) under nonreducing conditions. Indirect immunofluorescence microscopy demonstrated that three Fab clones stained the surfaces of late trophozoites/ schizonts and merozoites of the FCR3 and 3D7 strains, suggesting the Fabs' reactivities to a conserved epitope. Sequence analysis of the heavy-chain genes revealed that the closest germ line V segments were VH1-8 and VH7-81, with 91% to 98% homology. The closest germ line D segment was D3-10, and the closest germ line J segment was JH4 or JH5, with 90% to 97% homology. In the light-chain genes, the closest germ line V segment was A27 for the J kappa 2, J kappa 4, and J kappa 5 segments. The dissociation constants of these Fab fragments for recombinant MSP-1(19) ranged from 1.09 x 10(-9) to 2.66 x 10(-9) M. The binding of the three Fab fragments to MSP-I-19 was competitively inhibited by the anti-MSP-1(19) mouse monoclonal antibody 12.8, which inhibits erythrocyte invasion by merozoites. However, the human Fab fragment with the highest affinity did not inhibit in vitro growth of P. falciparum. This is the first report of gene analysis and bacterial expression of human monoclonal antibodies to P. falciparum MSP-1(19). The combinatorial immunoglobulin gene library derived from malaria patients provides a potential tool for producing high-affinity human antibodies specific for P. falciparum.