| Project/Area Number |
61570250
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Immunology
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| Research Institution | Aichi Cancer Center Research Institute |
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Principal Investigator |
SHIMADA Koichiro Laboratory of Biochemistry, 生化学部, 主任研究員 (40073126)
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| Co-Investigator(Kenkyū-buntansha) |
NISHIDA Yasuyoshi Molecular Biology Unit, 分子生物学研究室, 室長 (50107059)
NIKAIDO Toshio Molecular Biology Unit, 分子生物学研究室, 研究員 (50180568)
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| Project Period (FY) |
1986 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1987: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1986: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | Nuclear antigen / Gene cloning / 抗核抗体 |
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| Research Abstract |
Using sera of patients with systemic lupus erythematosus, we have isolated from bovine retina cDNA library a clone that produces a fusion protein immunologically reactive one of the antibodies in the sera.Using this cDNA as a probe,we have also obtained a homologous human placenta cDNA colne covering a full portion of coding region.Nucleotide sequences of the cDNAs and its predicted amino acid sequences of an poen reading frame were determined.No significant homologous sequences to these cDNAs were not detected in the sequences enlisted in the Data-Base of GenBank and NBRF.Comparable amino acid sequences in these human and bovine cDNA clones were exactly the same except one amino acid exchange.Such a strict conservation in evolution argues for the importance of this new gene in normal cell physiology.We have purified the fusion protein from the extract of E.coli carring expression recombinant of the bovine cDNA clone.
Polyclonal antisera to this fusion protein were generated in rabbits
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and monoclonal antibody with mouse hybridoma cells.Nuclei of the cultured cells were specifically stained with these antibodies and a nucoear localization signal amino acid sequence was recognized in the predicted open-reading frame.These findings suggest that this gene product may be one of the nuclear antigens. Autoimmune sera (250 samples) were screened against the purified fusion protein by using a solid-phase microtiter ELISA.It was found that the titer distribution for this fusion protein was quite similar to that of the Ki antigen already known as one of the nuclear antigens.
already known as one of the nuclear antigens.
The fusion protein and Ki antigen (kindly given by Dr Y.Takasaki) were examined by western immunoblots to show that both main bands have a quite similar mobility on SDS-PAGE and share common antigenic epitope(s).
In summary isolate cDNA clones seem to be the gene of Ki antigen or analogous gene(s) of nuclear antigen sharing cross-reactive immunological epitope(s). Cloning of the Ki antigen has not been reported. Less
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