2005 Fiscal Year Final Research Report Summary
Development of rapid screening and production technique of antibody Fv for protein chip
| Project/Area Number |
13854003
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| Research Category |
Grant-in-Aid for Scientific Research (S)
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| Allocation Type | Single-year Grants |
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| Research Field |
生物・生体工学
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| Research Institution | The University of Tokyo |
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Principal Investigator |
NAGAMUNE Teruyuki The University of Tokyo, Graduate School of Engineering, Department of Chemistry and Biotechnology, Professor, 大学院・工学研究科, 教授 (20124373)
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| Co-Investigator(Kenkyū-buntansha) |
UEDA Hiroshi The University of Tokyo, Graduate School of Engineering, Department of Chemistry and Biotechnology, Associate Professor, 大学院・工学研究科, 助教授 (60232758)
SINKAI Msashige The University of Tokyo, Graduate School of Engineering, Department of Chemistry and Biotechnology, Lecturer, 大学院・工学研究科, 講師 (70262889)
KAWAHARA Masahiro The University of Tokyo, Graduate School of Engineering, Department of Chemistry and Biotechnology, Assistant Professor, 大学院・工学研究科, 助手 (50345097)
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| Project Period (FY) |
2001 – 2005
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| Keywords | Chimeric Receptor / Antigen concentration dependent cellular growth / Library screening / Antibody variable region secretive cell / Electrospray deposition method / Antibody microarray |
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| Research Abstract |
We constructed HE and LE chimeric receptors comprising V_H and V_L of anti-hen egg lysozyme (HEL) antibody fused with extracellular D2 and transmembrane/intracellular domains of erythropoietin receptor, respectively. H_g and L_g chimeric receptors were also constructed by replacing intracellular domain with that of gp130. Furthermore, a series of chimeric receptors whose ligand recognition domain is anti-fluorescein antibody single chain F_v were constructed. After the gene transduction, genetically modified cells were successfully amplified by addition of cognate antigen in the culture medium.
Next, we randomized 4 amino acids of HEL-recognition site in V_H, linked to gp130, and transduced the resultant vector into LE-expressing cells. After HEL selection, the mutant V_H showed comparable binding affinity to the wild-type V_H. Furthermore, V_H library was amplified by PCR from splenocyte of mice immunized with superoxide dismutase (SOD), and fused with gp130 intracellular domain. When cells were transduced with this V_H library, some growing clones were obtained, indicating the possibility of library selection by using the chimeric receptor.
In addition, we constructed a vector in which chimeric receptor gene is flanked with two loxP sequences. By using this vector, we successfully developed a system in which a chimeric receptor-expressing cell can be sequentially converted into an antibody producer cell, which was attained by expressing Cre recombinase after antigen selection in the cell. Furthermore, we developed a technique to make 324 homogeneous spots of antibody at 150 μm diameter on a glass plate (9mm x 9mm) by an electrospray deposition method. Sandwich ELISA using this antibody microarray reproducibly detected antigen concentration at 0.1 to 1 ng/ml of detection limit.
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Research Products
(43 results)
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[Journal Article] Exogenous gene expression and growth regulation of hematopoietic cells via a novel human artificial chromosome.2006
Author(s)
Yamada, H., Kunisato, A., Kawahara, M., Tahimic, C.G.T., Ren, X., Ueda, H., Nagamune, T., Katoh, M., Inoue, T., Nishikawa, M., Oshimura, M.
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Journal Title
J. Hum. Genet. 51
Pages: 147-150
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Reversal of antigen-dependent signaling by two mutations in antibody/receptor chimera : implication of inverse agonism in cytokine receptor superfamily.2004
Author(s)
Kawahara, M., Ishii, S., Tsumoto, K., Kumagai, I., Ueda, H., Nagamune, T.
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Journal Title
Biochem. Pharmacol. 68
Pages: 539-548
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Antigen-mediated genetically modified cell amplification (AMEGA) with single vector transduction.2004
Author(s)
Kaneko, E., Kawahara, M., Tsumoto, K., Kumagai, I., Ueda, H., Nagamune, T.
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Journal Title
J. Chem. Eng. Jpn. 37
Pages: 1259-1264
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Cell proliferation switch responsive to fluorescein dimer.2004
Author(s)
Kimura, H., Kawahara, M., Tomlinson, I.M., Ueda, H., Nagamune, T.
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Journal Title
Animal Cell Technology : Basic ＆ Applied Aspects (Yagasaki, K., Ed.), 13., Kluwer Academic Publishers, Dordrecht
Pages: 335-339
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Bypassing antibiotic selection : positive screening of genetically modified cells with an antigen-dependent proliferation switch.2003
Author(s)
Kawahara, M., Ueda, H., Morita, S., Tsumoto, K., Kumagai, I., Nagamune, T.
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Journal Title
Nucleic Acids Res. 31
Pages: e32
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Fabrication of protein microarrays for immunoassay using the electrospray deposition method.2003
Author(s)
Lee, B.H., Kim, J., Ishimoto, K., Yamagata, Y., Tanioka, A., Nagamune, T.
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Journal Title
J. Chem. Eng. Jpn. 36
Pages: 1370-1375
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Selection of highly productive mammalian cells based on an inducible growth advantage using an antibody/receptor chimera.2002
Author(s)
Kawahara, M., Ueda, H., Tsumoto, K., Kumagai, I., Mahoney, W., Nagamune, T.
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Journal Title
J. Biosci. Bioeng. 93
Pages: 399-404
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Antigen-mediated genetically modified cell amplification2002
Author(s)
Kawahara, M., Ueda, H., Tsumoto, K., Kumagai, I., Mahoney, W., Nagamune, T.
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Journal Title
ACS SYMPOSIUM SERIES 830 (Biological System Engineering)
Pages: 140-152
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Lysozyme-dependent proliferation switch : Hematopoietic and hybridoma cell growth control with the use of antibody/gp 130 chimera.2002
Author(s)
Kawahara, M., Natsume, A., Terada, S., Kato, K., Tsumoto, K., Kumagai, I., Miki, M., Mahoney, W., Ueda, H., Nagamune, T.
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Journal Title
Animal cell technology for creation of new era (JAACT 2000), Kluwer Academic Publishers, Dordrecht
Pages: 337-341
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] A growth signal with an artificially induced erythropoietin receptor-gp 130 cytoplasmic domain heterodimer2001
Author(s)
Kawahara, M., Ueda, H., Tsumoto, K., Kumagai, I., Manohey, W., Nagamune, T.
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Journal Title
J. Biochem. 130
Pages: 305-312
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Replacing factor dependency with that for lysozyme : Affordable culture of IL-6 dependent hybridoma by transfecting artificial cell surface receptor.2001
Author(s)
Kawahara, M., Natsume, A., Terada, S., Kato, K., Tsumoto, K., Kumagai, I., Miki, M., Mahoney, W., Ueda, H., Nagamune, T.
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Journal Title
Biotechnol. Bioeng. 74
Pages: 416-423
Description
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