| Co-Investigator(Kenkyū-buntansha) |
ASANO Ryutaro TOHOKU UNIVERSITY, GRADUATE SCHOOL OF ENGINEERING, ASSISTANT PROFESSOR, 大学院・工学研究科, 助手 (80323103)
UMETSU Mitsuo TOHOKU UNIVERSITY, IMIRM, ASSISTANT PROFESSOR, 多元物質科学研究所, 助手 (70333846)
TSUMOTO Kouhei TOHOKU UNIVERSITY, GRADUATE SCHOOL OF ENGINEERING, ASSOCIATE PROFESSOR, 大学院・工学研究科, 助教授 (90271866)
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| Research Abstract |
A major goal of this study is construction of novel preparation system of human antibody molecules based on human antibody genome information by diversity creation of genes, in vitro stable selection via recognition mechanism, and development of preparation system of selected molecules. The results are summarized as follows.
1.Diversity of genes:
DNA primers for specific amplification of human and mouse antibody variable domain genes have been designed, and the experimental conditions have been established. In addition to overlap-extension PCR, experimental conditions for error-prone PCR for randomization of antibody variable domain genes, revealing that concentration of metal ions and control of reaction time are critical for efficient amplification and mutation of the genes.
2.Selection:
Novel selection method on the basis of Fv stabilization under existence of antigens has been established. Using this method, human antibody molecules specific for EGF receptor, NK cell inhibitory recepto
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r, bio-degradable plastic surface, and gold could be selected. A humanized antibody could be functionally improved via random mutation under conditions described in 1 and selection of the phage-displayed phages using target-expressed cells. Selection system using a SPR instrument has been constructed.
3.Preparation system:
For refolding of antibodies and cytokines, their refolding procedure has been improved, and spectroscopic evidence for high refolding efficiency of the additive-induced step-wise dialysis method has been elucidated. Preparation of Fv-fusion protein, e.g. alkaline phosphatase and cytokines, has been established and recombinant bispecific antibody, diabody has been also constructed.
4.Molecular recognition:
Human antibody variable regions, obtained from selection in 2, have been prepared using E. coli expression system, and functional characterization using ITC, QCM and/or SPR has been performed. Selected Fv fragments have also been characterized from structural viewpoints, revealing several novel insights into the mechanism of antibodies for antigen recognition. Less
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