| Project/Area Number |
07457420
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
小児外科
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| Research Institution | UNIVERSITY OF TOKYO |
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Principal Investigator |
KAMII Yoshiyuki (1996) University of Tokyo, Faculty of Medicine, Instructor, 医学部・附属病院, 助手 (70177567)
土田 嘉昭 (1995) 東京大学, 医学部(病), 教授 (80010164)
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| Co-Investigator(Kenkyū-buntansha) |
OBANA Kazuko University of Tokyo, Faculty of Medicine, Instructor, 医学部・附属病院, 助手 (60272580)
TSUCHIDA Yoshiaki Gunma Children's Medical Center, Director, 院長 (80010164)
逸見 仁道 東邦大学, 医学部, 助教授 (90165514)
上井 義之 東京大学, 医学部(病), 助手 (70177567)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1996: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | Neuroblastoma / N-Myc protein / ELISA / MAP method / recombinant N-Myc / Peptide antibody / N-myc / c-myc / ペプチド抗体 |
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| Research Abstract |
The importance of determining N-Myc oncoprotein rather than genomic N-myc amplification has been emphasized in neuroblastoma. In order to develop an ELISA method for N-Myc oncoprotein quantification, an effort was made to raise antibodies specific for N-Myc, and also to produce a relatively smaller-sized N-Myc oncoprotein, which should be water soluble. N-Myc-specific peptide (codon.223-239) were synthesized and injected into rabbits in conjugation with lysine core (MAP method) plus adjuvant. Synthesized peptide conjugated to the lysine core raised a potent antibody. Both crude antibody and IgC purified on an affinity column on such peptide coupled to EAH Sepharose showed a precipitation line identical to that of N-Myc oncoprotein by immunoblot analysis. The purified IgG against N-Myc-specific peptide (codon.223-239) strongly stained the nuclei of neuroblastoma cells with N-myc amplification ; thus a polyclonal anti-body specific for a synthetic peptide from the N-Myc oncoprotein was obtained.
For preparing standard protein, partial exon 2 and exon 3 of the N-myc gene was cloned and inserted into an expression vector, pET16b. A water-soluble recombinant N-Myc oncoprotein (rN-Myc) with a molecular weight of 38 kDa was expressed by the Escherichia coli, and was purified with Ni^<2+> affinity column chromatography. In immunoblot analysis, the purified anti-N-Myc-specific peptide (codon.223-239) IgG reacted with the rN-Myc oncoprotein. Thus, a water-soluble rN-Myc was successfully obtained, which will enable the establishment of a quantitative method for measuring N-Myc oncoprotein expression.
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