| 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)
神谷 典穂 東京大学, 大学院・工学系研究科, 助手 (50302766)
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| Project Period (FY) |
2001 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥118,040,000 (Direct Cost: ¥90,800,000、Indirect Cost: ¥27,240,000)
Fiscal Year 2005: ¥11,830,000 (Direct Cost: ¥9,100,000、Indirect Cost: ¥2,730,000)
Fiscal Year 2004: ¥11,700,000 (Direct Cost: ¥9,000,000、Indirect Cost: ¥2,700,000)
Fiscal Year 2003: ¥11,700,000 (Direct Cost: ¥9,000,000、Indirect Cost: ¥2,700,000)
Fiscal Year 2002: ¥34,970,000 (Direct Cost: ¥26,900,000、Indirect Cost: ¥8,070,000)
Fiscal Year 2001: ¥47,840,000 (Direct Cost: ¥36,800,000、Indirect Cost: ¥11,040,000)
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| Keywords | Chimeric Receptor / Antigen concentration dependent cellular growth / Library screening / Antibody variable region secretive cell / Electrospray deposition method / Antibody microarray / 抗体 / 受容体 / ライブラリー / 抗体生産 / プロテインチップ |
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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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