| 研究課題/領域番号 |
16F16404
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| 研究機関 | 国立研究開発法人理化学研究所 |
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研究代表者 |
間 陽子 国立研究開発法人理化学研究所, 伊藤ナノ医工学研究室, 研究員 (50182994)
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| 研究分担者 |
BAI LANLAN 国立研究開発法人理化学研究所, 伊藤ナノ医工学研究室, 外国人特別研究員
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| 研究期間 (年度) |
2016-10-07 – 2019-03-31
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| キーワード | bovine leukemia virus / CAT1/SLC7a1 / BLV receptor / common B cell epitope / receptor binding domain |
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| 研究実績の概要 |
The CHO cells do not express functional entry receptor for BLV. The CAT1/SLC7a1 as a receptor for BLV was identified. Therefore, the fragment of CAT1/SLC7a1 gene was amplified from CD5+ B cell of natural host of BLV, KU-1, and inserted into mammalian expression vector and then transfected into CHO cells to express BLV receptor. The transfected CHO cells was co-cultured with FLK-BLV cells and then can detect syncytium formation. For determining BLV receptor binding domain, the common B cell epitope was inserted into Escherichia coli (E.coli.) expression vector pGEX that GST-tag was replace with His-tag. The common B cell epitope fusion protein expressed and purified from E.coli. Now I am determining CAT1/SLC7a1 binding domain using purified fusion protein by pull down assay.
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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
For determining the BLV receptor binding domain, the common B cell epitope was inserted into E.coli expression vector pGEX that GST-tag was replace with His-tag. The fusion protein was expressed and purified in E.coli. The CHO cells do not express functional entry receptor for BLV. The CAT1/SLC7a1 as a receptor for BLV was identified. Therefore, the fragment of CAT1/SLC7a1 gene was amplified from CD5+ B cell of natural host of BLV, KU-1 and inserted into mammalian expression vector, and then transfected into CHO cells to express BLV receptor. The transfected CHO cells was co-cultured with FLK-BLV cells and then can detect syncytium formation. The CAT1 /SLC7a1 was purified. Now I am performing determination of CAT1/SLC7a1 binding domain using purified fusion protein by pull down assay.
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| 今後の研究の推進方策 |
CC81 cell can infect BLV and form syncytia, indicating CC81 cell expresses receptor for BLV. So CAT1/SLC7a1 will be knockdown in CC81 cell and co-culture with free BLV or BLV infected cell to measure whether BLV another receptor present or not by syncytium assay. If CC81 cell expresses another receptor for BLV, the receptor will be cloned and then analyze the sequences of cDNA. The next, the function of receptor which the virus binding ability of receptor and cell fusion will be detected by syncytium assay. The other hand, the CAT1/SLC7a1 was purified to determine the binding domain using common B cell epitope fusion protein by pull-down assay. Whether common B cell epitope binds to CAT1/SLC7a1 or not will be confirmed by neutralization assay.
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