1996 Fiscal Year Final Research Report Summary
Quantification of myc gene products and its clinical implication
| 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 University of Tokyo, Faculty of Medicine, Instructor, 医学部・附属病院, 助手 (70177567)
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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)
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| Project Period (FY) |
1995 – 1996
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| Keywords | Neuroblastoma / N-Myc protein / ELISA / MAP method / recombinant N-Myc / Peptide antibody |
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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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