| Project/Area Number |
16580163
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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 |
General fisheries
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| Research Institution | National Research Institute of Aquaculture, Fisheries Agency |
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Principal Investigator |
GEN Koichiro National Research Institute of Aquaculture Fisheries Agency, Aquaculture Biology Division, Senior Researcher, 養殖研究所・生産技術部, 主任研究員 (80372051)
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| Co-Investigator(Kenkyū-buntansha) |
KAGAWA Hirohiko Miyazaki University, Department of Biological Production and Environmental Science, Professor, 農学部生物環境科学科, 教授 (60169381)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2005: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2004: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | gonadotropins / baculovirus expression system / recombinant protein / red seabream / steroidogenesis / バキュロウイルス共感染系 / 雄性ホルモン産生能 / 雌性ホルモン産生能 / 共感染系 / 共発現系 |
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| Research Abstract |
Recent advances in recombinant gene technology have enabled the production of biologically active fish gonadotropins (GTHs : FSH and LH). However, an efficient expression method for the production of GTHs has not yet been developed. In this study, we attempted to produce red seabream GTHs in silkworm larvae using two different baculovirus expression systems. To synthesize recombinant red seabream heterodimeric GTHs, two strategies were employed. The first utilized a dual vector expression strategy, in which each GTH subunit (α, FSHβ and LHβ) was expressed from a separate comparative vector in a single host. The second method used a bicistronic expression strategy, in which both GTH subunit cDNAs were expressed from the vector. Either a dual or a bicistronic expression vector inoculated into silkworm larvae of the 5th instar early stage. Five days after inoculation, the hemolymph of the larvae containing recombinant FSH and LH was collected, and examined of biochemical property and biological activity. Western blot analysis revealed that by using a dual vector expression system, each subunit of FSH and LH was successfully synthesized and secreted into the hemolymph, allowing the synthesis of heterodimeric molecules. In the case of the bicistronic vector coexpressing GTH subunits, the yields of FSHα and LHα subunits was very low, whereas those of both FSHβ and LHβ subunits were high. The biological activities of GTHs produced by a dual vector system were comparable to those of native GTHs in in vitro steroid hormone production. Both recombinant FSH and LH stimulated the in vitro 11-ketotestosterone production in sliced testis of red seabream. Furthermore, recombinant LH stimulated the in vitro production of estradiol-17β by ovarian follicles in a concentration-dependent manner. Conversely, GTHs derived from a bicistronic vector system had no effect on in vitro steroid hormone production.
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