| Project/Area Number |
06556015
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Tokyo University of Pharmacy and Life Science |
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Principal Investigator |
YAMAGATA Hideo Tokyo University of Pharmacy and Life Science, School of Life Sci., Professor, 生命科学部, 教授 (20023468)
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| Co-Investigator(Kenkyū-buntansha) |
IWASA Susumu Takeda Chemical Industries, Ltd., DDS Res. Lab., Group Leader, DDS研究所, 主席研究員
KAKINUMA Atsusi Nagoya University, School of Agriculture, Professor, 農学部, 教授 (50252276)
加藤 雅士 名古屋大学, 農学部, 助手 (70242849)
太田 敏博 東京薬科大学, 生命科学部, 助教授 (10266893)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥8,200,000 (Direct Cost: ¥8,200,000)
Fiscal Year 1995: ¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 1994: ¥4,600,000 (Direct Cost: ¥4,600,000)
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| Keywords | Bacillus brevis / Antibody fragments / Secretion / Urokinase / Expression vectors / Bacillus brevis / Fab′断片 / バチルス・ブレビス / 単鎖2価抗体 / Fab断片 / 血栓 / 血栓溶解剤 / リンカーペプチド |
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| Research Abstract |
Expression-secretion vectors for the production of anti-human urokinase antibody fragments, single chain (sc) Fv, Fab', and scFab', were constructed and inroduced into Bacillus brevis. The promoter of the cell wall protein gene operon (cwp), and the translation start site and the signal pepetide-encoding region from the mwp gene, the gene for one of the major cell wall proteins of B.brevis, were used in the vector. The cDNAs encoding the light and heavy chains of Fv and Fab' fragments were prepared by PCR from the cDNAs for mouse-human chimeric monoclonal antibody against urokinase-type plasminogen activator and inserted immediately downstream of the signal peptide-encoding region. To construct the genes for sc fragments, the cDNAs encoding the light and heavy chains were linked by synthetic oligonucleotides encoding flexible peptide linkers of 17 or 24 amino acids. Fab' was efficiently produced by B.brevis resulting in an accumulation at a level of 100 mg/I in the culture medium. In contrast, the amounts of scFv and scFab' produced by B.brevis were at a level of a few mg/I in the culture medium. Fab' produced by B.brevis was purified and confirmed to have an antigen binding activity comparable to that of the parental antibody.
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