| Project/Area Number |
02670166
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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 |
寄生虫学(含医用動物学）
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| Research Institution | Shinshu University |
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Principal Investigator |
SUGANE Kazuo Shinshu University School of Medicine, Professor, 医学部, 教授 (50112488)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUURA Tadashi Shinshu University School of Medicine, Assistant, 医学部, 助手 (60209492)
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| Project Period (FY) |
1990 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1991: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1990: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | Anisakis / specific antigen / cloning / gene / 翻訳産物 |
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| Research Abstract |
The gene encoding an antigenic polypeptide of Anisakis simplex larvae was studied using recombinant DNA techniques. cDNA synthesized from poly(A)-rich MRNA from A simplex larvae was ligated into phage vector lambda gtll DNA and packaged in vitro. The phages were propagated on Escherichia coli and a lambda gtll expression library was constructed. A cDNA clone encoding a 42 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method. A clone containing CDNA for a 42 kDa protein was isolated. The gene encoding this 42 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the CDNA as a probe. The gene was transcribed to MRNA with approximately 1400 nucleotides and translated to 42 kDa polypeptide. The antigenic beta-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera. An enzyme-linked immunosorbent assay (ELISA) using antigenic beta-galactosidase-cDNA fusion protein (FP) obtained by the recombinant DNA technique provided a useful diagnostic tool for anisakiasis. Anisakis-infected humans sera reacted strongly with FP that was immobilized with anti- beta-galactosidase monoclonal antibody on microplates. However, the FP did not react with sera from patients with other helminthiasis. In detection of anti-Anisakis IgG antibody, ELISA using FP was highly sensitive and specific compared to that using crude somatic antigen.
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