| Project/Area Number |
09670877
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Dermatology
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
TANAKA Toshihiro Kyoto Univ.Dermatology, Lecturer, 医学研究科, 講師 (50188314)
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| Co-Investigator(Kenkyū-buntansha) |
TODA Ken-ichi Kyoto Univ.Dermatology, Lecturer, 医学研究科, 講師 (80159045)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1997: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Keywords | bullous pemphigoid / hemidesmosome / pemphigoid antiger / BP180 / immunoglobulin / monoclonal antibody / immunoglobulin cDNA / basement menbrane / ヘミテマモゾーム / cDNA / 可変領域 / 塩基配列 / 人工免疫グロブリン / 実験水疱 |
|---|
| Research Abstract |
Bullous pemphigoid is a tissue specific autoimmune disease, characterized with clinical tense blister of the skin and histological subepidermal blistering. Patients have circulating autoantibodies against the component of basement membrane zone of the skin, named BP180 or pemphigoid antigen. We previously reported the establishment of a monoclonal antibody producing cell line. This monoclonal antibody induce subepidermal blister in neonatal mice. The purpose of this Grant-in aid for scientific research is, firstly, the molecular cloning of variable region of this monoclonal antibody and secondary, reconstruct this monoclonal antibody in mice to make an animal model. For this purpose, we fished immunoglobulin variable region cDNA by reverse transcriptase-polymerase chain reaction (RT-PCR) by using poly A RNA as a template and primers. Gel electrophoreseis of RT-PCR products revealed single band for heavy and light chain cDNA respectively with expected molecular weight. Each cDNA was sub
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cloned into sequencing vector. The nucleotide sequences of these heavy and light chain cDNA and deduced amino acid sequences of these molecules revealed that they have high homologies to other rat immunoglobuline and that they possess specific sequence at hyper variable regions. These cDNAs were reamplified with primers that contains 5 prime non-coding sequence and 3 prime splicing signals. An expression of immunoglobulin cDNA product was first confirmed by E.coli expression system. A crude extract of E.coli which was transfected with vector was applied to 12.5% SDS-polyacrylamide gel electrophoreses and immunoblot was performed with anti-tag antibody as first antibody. Western blot showed a single band of expected molecular weight, indicating that this cDNA do have an open reading frame and that the open reading frame is in frame to tag sequence. Function of reconstituted cDNA product was assayed by immunoflourescent study. Binding of fusion protein to basement membrane was detected by anti-tag antibody. In summary, experimental procedures are almost fully achieved. Less
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