2016 Fiscal Year Annual Research Report
共通Ｂ細胞エピトープを利用した新規牛白血病ウイルスレセプターの同定
| 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, 分子ウイルス学特別研究ユニット, ユニットリーダー (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, 分子ウイルス学特別研究ユニット, 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 / BLV / KU-1 / expression library / CDM8 vector / BLV receptor |
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| Outline of Annual Research Achievements |
The CD5+ B cell of natural hosts of BLV, KU-1 were grown and harvested. Then poly(A)+ RNA was prepared from KU-1 by oligo(dT) cellulose chromatography of total RNA isolated by the guanidinium thiocyanate method. The cDNA was synthesized by basically a variation on the method of Gubler and Hoffman and then cDNA fragments grouped into cDNA pools. The mammalian cell expression vector was constructed from CDM8 by inserting a synthetic transcription unit between the suppressor tRNA gene and the simian virus 40 (SV40) origin. The cDNA pools individually was ligated to the vector that be linearized by cleavage with a suitable restriction enzyme using non-selfcomplemently BstSI adaptors. The ligated DNA was transformed into competent cell Escherichia coli and then were prepared for paining.
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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
Previously identified common B cell epitope was used for screening the BLV receptor on cells which transfected cDNA library inserted expression vector. However, common B cell epitope is had to synthesize, and it is expensive, and easy to deteriorate that difficult to stock long time. so I successfully constructed the protein expression vector which was five interlocking common B cell epitope fragment inserted into pGEX GST-fusion protein expression vector. The expression vector was transformed into E.coli BL21 and then purified GST-common B cell epitope fusion protein for screening experiment.
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| Strategy for Future Research Activity |
The CHO cells do not express functional entry receptor for BLV. The cDNA inserted expression vector transfect into CHO cells to express BLV receptor and then will screen by previously identified common B cell epitope, gp51p16-C, or fusion protein. The gp51p16-C or fusion protein bound cell expressions as BLV receptor and to clone the BLV receptor in the productions of cell. The cDNAs from the gp51p16-C or fusion protein binding CHO cells will be extracted by Hirt extraction method and to get the clone of receptor only expressed cell and then isolate the cDNA to analyze the full sequences of cDNA by PCR method. A functional cell surface receptor is required for virus entry. So will detect function of receptor which the virus binding ability of receptor and cell fusion by syncytium assay.
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