| Project/Area Number |
11670432
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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 |
内科学一般
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
KOBAYASHI Seiichi Hokkaido Univ., Coll.of Med.Tech., Prof., 医療技術短期大学部, 教授 (30150246)
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| Co-Investigator(Kenkyū-buntansha) |
MORIYAMA Takanori Hokkaido Univ. Coll.of Med.Tech., Asso.Prof., 医療技術短期大学部, 助教授 (20312423)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2000: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1999: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Keywords | systemic lupus erythematosus / RT-PCR / alternative splicing / soluble CTLA-4 / gene transfer / monoclonal antibody / sandwich ELISA / carrier protein / 疾患活動性 / 二量体 / 可溶型CTLA-4 / 組換え蛋白 |
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| Research Abstract |
1.In the RT-PCR analysis of full-length CTLA-4 gene expression by PBMC from SLE patients, we cloned a CTLA-4 mRNA variant, which was generated by alternative splicing of the transmembrane-encoding exon 3. The 22 altered amino acid sequence in the C terminus was deduced in this CTLA-4 mRNA variant as well as the deletion of the transmembrane domain.
2.Culture supernatants from COS-7 cells transfected with the CTLA-4 variant cDNA-inserted expression vector were found to contain the soluble form of CTLA-4 (sCTLA-4), which indicated that the sCTLA-4 mRNA variant did encode a sCTLA-4 protein.
3.A monoclonal antibody, H11.6 (IgGa, κ), which reacts with sCTLA-4, was established by a standard cell fusion technique from the mice immunized with the synthetic peptide (the 22 amino acids above)-KLH conjugate in CFA and IFA.A newly-designed sandwich ELISA for sCTLA-4 was developed using commercially available anti-CTLA-4 mAb as a capture antibody and biotin-labeled H11.6 mAb as a detection antibody.
4.Serum sCTLA-4 levels in SLE patients were significantly lower than healthy controls (P<0.03). They, however, did not correlate with any laboratory data and a daily dosage of prednisolone.
5.sCTLA-4 gene-transfected COS-7 cells produced a 26kD protein in the culture supernatant. When it was mixed with human serum, sCTLA-4 activity was observed in identical fractions with serum albumin in both gel fractionation HPLC and ion exchange chromatography. These data strongly suggested that serum albumin is one of the carrier proteins for sCTLA-4.
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