| 研究課題/領域番号 |
26870872
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| 研究機関 | 長崎大学 |
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研究代表者 |
モイ メンリン 長崎大学, 熱帯医学研究所, 准教授 (40597499)
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| 研究期間 (年度) |
2014-04-01 – 2017-03-31
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| キーワード | dengue / ADE / dengue hemorrhagic fever |
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| 研究実績の概要 |
To study the role of FcγR in infection-enhancement (ADE) of dengue virus (DENV) during infection, we focused on determination of the types of FcγR and receptor cytoplasmic regions which are involved in ADE. In FYI 2014, using molecular techniques, FcγRI, γ-chain and FcγRIIA genes were constructed and sub-clonned into mammalian expression vectors with antibiotic resistant cassettes to construct the FcγRIIA-pcDNA3.1(neo), FcγRIA-pCMV6A(bsd), and γ-chain-pCMV6A(puro) plasmids. The plasmids were then transfected into a baby hamster kidney cell (BHK) cell line using transfection reagents, and cell surface expression of FcγRIIA and FcγRIA, and expression of γ-chain in the intercellular compartment in each of the cell lines were confirmed using flow-cytometry and western blot. Because FcγRIA requires an activation chain (γ-chain) to function, dual transfection of FcγRIA-pCMV6A(bsd) and γ-chain-pCMV6A(puro) plasmids into BHK cells was also performed. Antibiotic-resistant cells were then selected and were further selected for resistant colonies using the limited-dilution method. Resistant colonies were then selected using protein expression as a criteria by flow-cytometry. Four cell lines stably expressing the FcγRIA, FcγRIIA, γ-chain and the FcγRIA-γ chain were successfully established and propagated.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
FcγR-expressing plasmids were constructed and protein expression of each of the receptors were confirmed. Cell lines stably expressing the receptors were successfully generated. The cell lines would prove useful for further studies on ADE in DENV infection.
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| 今後の研究の推進方策 |
Constructs of the FcγR with altered cytoplasmic regions would be generated and the role of each receptor in ADE will be determined using these cell lines by DENV infection assay. In addition, using the stable cell lines developed in the previous year, cellular factors and markers involved in ADE antibody immune-complex internalization via the FcγR will be determined, to establish fundamental knowledge and novel approaches for DENV therapeutics and clinical applications.
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| 次年度使用額が生じた理由 |
(1) Travel expenses planned for research presentation were partially covered by another funding.
(2) Lesser consumables were purchased for gene construction and transfection because the experiments went well.
(3) Due to importation/manufacturer issues, purchases of consumables (reagents such as antibodies for receptor detection) planned for the previous year will be carried forward to the next financial year.
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| 次年度使用額の使用計画 |
Incurring amount will be used for:
1. Tools and consumables for cell culture and molecular (protein and genomic) studies. 2. Travel expenses for participation and presentation in local and international meetings and conferences, to (a) disseminate research achievements to the general public, (b) promote information exchange, and (c) gather new information for further research advancement. 3. Publication fees and proofreading to publish findings and disseminate results to society and citizens.
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