| Project/Area Number |
16590977
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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 |
膠原病・アレルギー・感染症内科学
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| Research Institution | The University of Tokyo |
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Principal Investigator |
KAWASAKI Hiroshi The University of Tokyo, Institute of Medical Science, Research Associate, 医科学研究所, 助手 (80280957)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2005: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2004: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | Cytokine / Receptor / Autoimmunity / Acquired Immunity / Innate Immunity / Tumor Immunity |
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| Research Abstract |
We established a panel of monoclonal antibodies raised against human IL-12R beta1 chain. In the course of characterizing them, a molecule that coprecipitate with UL-12R beta1 chain came to our notice. Later, the IL-23R was cloned and characterized, which revealed that IL-23R was composed of IL-12R beta1 chain and IL-23-specific IL-23R beta2 chain. We predicted that the production of the monoclonal antibodies against IL-23R beta1 chain would contribute to the elucidation of the mechanisms involved in the induction of acquired immunity initiated by IL-12 and IL-12R. The strategy we picked up was to immunize mice with tumor cell line expressing the IL-23R beta2 chain. In order to express it, we took advantage of the signal-trap method utilizing CD25 signal peptide sequence that has SacI site at the junction of signal sequence and coding sequence. We subcloned the cDNA of the IL-23R beta1 chain extracellular domain into this SacI site and transfected and expressed in a murine B cell line under the control of SR alpha promoter. The expression of this chimeric gene product was confirmed by the staining of CD25 in flowcytometric analysis.
The mmurine cell that has the highest expression of the chimeric protein was used for the immunogen and the immunize mice were sacrificed for cell fusion using the non-productive myeloma cell line with polyethylene glycol method. So far, successful fusion is still in vain. The problem involved is that the chimeric molecule is labile to degradation, which was guessed by the CD25 reactivity of the supernatant of the cells rather than the lysate of cells. We are in the process of using other tag protein including GFP and c-myc.
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