2017 Fiscal Year Annual Research Report
Identification of novel receptor for bovine leukemia virus via cloning with common B cell epitope
| Project/Area Number |
16F16404
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
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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| Co-Investigator(Kenkyū-buntansha) |
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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| Project Period (FY) |
2016-10-07 – 2019-03-31
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| Keywords | bovine leukemia virus / CAT1/SLC7a1 / BLV receptor / common B cell epitope / receptor binding domain |
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| Outline of Annual Research Achievements |
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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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
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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| Strategy for Future Research Activity |
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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[Presentation] A Vaccine Targeting Bovine Leukemia Virus Susceptible Cattle Suppresses Proviral Load2017
Author(s)
Yoko Aida, Lanlan Bai, Jiyun Kim, Pan He, Yuki Matsumoto, Noriaki Okimoto, Junya Yamagishi, Seiichi Tada, Yoshihiro Ito, Junko Kohara, Shin-nosuke Takeshima
Organizer
18th International Conference on Human Retrovirology HTLV and Related Viruses
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[Presentation] Development of a bovine leukemia virus-like particle vaccine and its immunogenicity in mice and cattle2017
Author(s)
Hiroyuki Otsuke, Saeki Negishi, LanLan Bai, Hirotaka Sato, Shin-nosuke Takeshima, Yoshitaka Imaizumi, Yasuko Nagai, Atsushi Iwamoto, Taichi Noro, Satoshi Sakamoto, Yuki Yamaguchi, Hiroshi Handa, Eiji Oishi,Yoko Aida
Organizer
18th International Conference on Human Retrovirology HTLV and Related Viruses
