| Project/Area Number |
01570224
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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 | Jikei University School of Medicine |
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Principal Investigator |
MAKIOKA Asao Jikei University School of Medicine, Parasitology, Lecturer, 医学部, 講師 (90119850)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1990: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1989: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Genetic engineering / Toxoplasma gondii / Major membrane antigen / PCR / Expression vectors / Fusion protein / Macrophage activation / マクロアァ-ジ活性化 / トキソプラズマ主要膜抗原 |
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| Research Abstract |
We have examined expression in Escherichia coli of the gene encoding the major surface antigen (P30) of Toxoplasma gondii. For this, the coding sequence of P30 was amplified by polymerase chain reaction (PCR) and cloned into two kinds of plasmid expression vectors. The recombinant plasmids were then transfected into E. Coli. SDS-polyacrylamide gel electrophoresis and immunoblot analysis of total cellular proteins from E. Coli transformed with parental or recombinant plasmids were performed to detect the product of P30 gene. No production of the P30 protein was observed with the vectors used for direct expression. On the other hand, the P30 gene cloned into the plasmid, pGEX-1 was highly expressed as a glutathione S-transferase (GST ; EC 2.5.1.18) fusion protein. The fusion protein was present in the pellet after cell lysis and its purification with affinity absorption on immobilized glutathione was unsuccessful because of its insolubility. The fusion protein as an aggregate was isolated from cell lysis pellet by urea washing procedure that yields nearly homogeneous fusion protein. Next we have performed experiments to determine whether immunization of mice with the P30 fusion protein activates macrophages to be able to kill T. Gondii in vitro. Inoculation of mice with 50 mug of the fusion protein caused significant activation of the macrophages. Marked activation was elicited when mice were injected with 100 mug or more of the fusion protein. On the other hand, no activation of the macrophages was observed even when mice were injected with 200 mug of GST protein, suggesting that the activation of macrophages is due to P30 in the fusion protein not to GST.
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