| Project/Area Number |
10670236
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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 |
寄生虫学(含医用動物学)
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| Research Institution | Keio University |
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Principal Investigator |
ASAI Takashi Keio University, School of Medicine, Department of Tropical Medicine and Parasitology, Assistant Professor., 医学部, 講師 (50175163)
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| Co-Investigator(Kenkyū-buntansha) |
NOZAKI Tomoyoshi Department of Parasitology, National Institute of Infections Diseases, Head of Molecular Parasitology., 室長 (60198588)
SANUKI Junichi Keio University, Instructor, 医学部, 助手 (90255571)
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2000: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1999: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | Toxoplasma gondii / Neospora caninum / NTPase / Gene / ATPase / Serodiagnosis / Parasitic Diseases / トキソプラズマ |
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| Research Abstract |
We have identified type I nucleoside triphosphate hydrolase (NTPase ; EC 3.6.1.3) activity, previously thought to be restricted to the virulent strains of Toxoplasma gondii, in the cell extracts of Neospora caninum tachyzoites. Sequence analysis of a complete cDNA from Nc-1 strain indicated that the N.caninum NTPase gene (NcNTPase) shared approximately 69% identity to the NTPases of T.gondii and is most similar to the NTPase-I isozyme. Southern blot analysis of genomic DNA and sequence analysis of two independent NcNTPase clones from Nc-1 strain revealed the presence of multiple genes, at least two of which are transcribed. Substrate specificity and Km values for ATP and ADP hydrolysis for recombinant or partially-purified native NcNTPase were the same as the type-I isozyme (NT Pase-I) found in type I virulent strains of T.gondii. NcNTPase was expressed in Escherichiacoli as inclusion bodies and purified after refolding. The purified NcNTPase was tested for their usefulness as antigen for the serodiagnosis of acute N.caninum infection in immunocompetent humans. The test was conducted by using the recombinant NcNTPase and comparing the enzyme linked immunosorbent assay (ELISA) absorbances with the Sabin-Feldman dye test titer. The total positive rate in dye test positive sera was very low for refolded recombinant NcNTPase. There were some positive sera for this recombinant NcNTPase, however, these sera did not react to other N.canium antigens and reacted to T.gondii antigens, indicating that they were toxoplasmosis sera. The data suggested that the recombinant NcNTPase was an useful antigen for serodiagnosis of N.caninum infection.
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