Preparation And Characterization Of Recombinant Human Monoclonal Antibody Fab Fragment Specific For Plasmodium Falciparum

Objection:This study is to prepare anti-Plasmodium falciparum human antibody Fab fragment by using a combinatorial immumoglobulin gene library,and to provide theoretic and experimental foundation for developing therapeutic antibody.Method:A combinatorial immunoglobulin gene library was constructed from peripheral lymphocytes of 8 patients with falciparum malaria.The C-terminal portion of the Plasmodium falciparum merozoite surface protein(MSP1₁₉) is a potential vaccine against erythrocytic stages of malaria.We used recombinant MSP1₁₉ as antigen and screened the library by colony blotting,enzyme-linked immunsorbent assay and immunoflurescent assay.Positive clones were sequenced and analyzed with the IgBlast program.The positive clones were purified by affinity chromatography from large scale of bacterial cultures.The affinity constant of Fabs was assessed by surface plasmon resonance using the BIAcore3000 instrument.Effect of human Fab on the growth of P.falciparum was examined in vitro.Site-directed mutagenesis in the third complementarity-determining region(CDR3) was used to enhance the affinity of the human Fab fragment.Result:The combinatorial immunoglobulin gene library contained approximately 5×10⁷ clones.The recombinant MSP1₁₉ was of antigen-speciality and good purity. When 8×10⁵ clones were screened,only two clones,designated as Pf25 and Pf227, were reacted specifically with the recombinant MSP1₁₉ and native protein of Plasmodium falciparum merozoite.The sequence homology of these clones with germlines was analyzed by IgBLAST.For Pf25,the closest V-segment germline of the heavy chain genes was VH1-8(97%homology),and that of the light chain gene was A27(100%homology).For Pf227,the closest V-segment germline of the heavy chain genes was VH1-8(98%homology),and that of the light chain gene was A27(99% homology).Affinity of Pf25 and Pf227 to recombinant MSP1₁₉ was measured by surface plasmon resonance;dissociation constants were 1.09×10^-9M and 2.66×10^-9 M respectively.Affinity of Pf25 is approximately 3 times higher than Pf227.In growth inhibition assay in vitro,no significance difference of parasitemia was detected between Pf25 and control human Fab.Site-directed mutagenesis in the CDR3 of Pf25 could get new clones with higher affininty.Conclusion:The important observation in this study was identification of primary structure of anti-MSP1₁₉ human antibodies.Molecular basis of immunoglobulin to MSP1₁₉,such as usage of germlines,and increasing the affinity of Fab fragment by site-directed mutagenesis in the CDR3 of L chain and H chain,were clarified first time,to our knowledge.