Construction of libraries of artificial antibodies and their databases
| Project/Area Number |
06557026
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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 |
Immunology
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| Research Institution | Institute for Comprehensive Medical Science, Fujita Health University |
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Principal Investigator |
KUROSAWA Yoshikazu Inst.Comp.Med.Sci, Fujita Health Univ. Professor, 総合医科学研究所, 教授 (10109259)
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| Co-Investigator(Kenkyū-buntansha) |
IBA Yoshitaka Inst, Comp.Med.Sci.Fujita Health Univ.Research Associate, 総合医科学研究所, 研究員
ITO Wataru Inst.Comp.Med.Sci, Fujita Health Univ.Assistant Professor, 総合医科学研究所, 講師 (50192498)
安井 久司 藤田保健衛生大学, 総医研, 助手 (60220141)
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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 |
¥20,700,000 (Direct Cost: ¥20,700,000)
Fiscal Year 1995: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1994: ¥18,000,000 (Direct Cost: ¥18,000,000)
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| Keywords | artificial antibody / phage antibody / affinity to antigen / antigen antibody interaction / polymerase change interuction / comprementarity-determining reagen / 抗体ライブラリー / PCR / CDR / カロリメーター / 抗HEL抗体 / protein A融合タンパク / データベース / コンピュータネットワーク |
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| Research Abstract |
This project attempted to construct libraries of artificial antibodies which contain antibodies selectively bound to any kind of antigens. Antibodies are expressed on the surface of M13 phages as an Fab form fused with cplll molecules. After isolation of phage antibodies, Fab portions can easily be converted to the form fused with protein A in our gene construct. To make libraries, while the sequences of the framework in V regions are kept constant, only the sequences of the comprementarity-determining regions (CDR) that form antigenbinding sites are highly diverged by use of polymerase chain reaction (PCR). In the present study, we constructed a library composed of 3x10^8 independent clones and analyzed the characteristics of clones in the library. The 20 clones randomly isolated from the library without screening showed the highly diverged sequences in the CDR as expected. The phage antibodies bound to hen egg white lysozyme (HEL) were isolated by panning method. Binding of phages to HEL-recovery of the bound phage-growth of phages, one round of this process requires two days. After three rounds of the panning, 20 clones were isolated from the recovered phages. 19 of them had anti-HEL activity and the binding constnats distributed from 10^6 to 10^7 M^<-1> The suquences of the CDR of antibodies that had anti-HEL activities indicated that more than half of them were unique and the rest had diverged residues. This suggested that some residues are required for showing anti-HEL activities but that some residues are not essential. Since the library that were constructed in the present study was diverged based on the sequence of anti-HEL antibody, D1.3, the antigens that could be covered by this library may have been biassed. Now, we are performing the grafting experiments of the CDR seuquences of various antigen-specific antibodies and are going to diversity the CDR sequences using the grafted clones as templates.
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Report
(3 results)
Research Products
(15 results)
