| 研究実績の概要 |
CAT1/SLC7A1 has been identified as a candidate receptor for bovine leukemia virus (BLV), but whether it is actual receptor remains unknown. BLV binds to cellular receptors on target cells, its membrane fusion is activated, and the virus enters the host cells. The CHO-K1 cells do not express detectable CAT1/SLC7A1 protein and resistant to BLV infection. Therefore, I amplified the CAT1/SLC7A1 gene from cDNA of CD5+ B cell of natural host of BLV and inserted into mammalian expression vector. The large syncytia were detected in CAT1/SLC7A1 transfected CHO-K1 cells that were co-cultured with permanently BLV-infected FLK-BLV cells or the culture supernatant of FLK-BLV cells. These results clearly indicated that bovine CAT1/SLC7A1 expression in resistant CHO-K1 cells conferred susceptible to BLV infection. Next, to further determine whether CAT1/SLC7A1 acted as a cell surface receptor for BLV infection, we verified that BLV particles bound to CAT1/SLC7A1 using flow cytometry. The result showed that CAT1/SLC7A1 functioned as a BLV attachment receptor. Third, for determining CAT1/SLC7A1 binding domain, the common B cell epitope was amplified and inserted into Escherichia coli expression vector pGEX that GST-tag was replace with His-tag to express and purified the fusion protein. I tried to determine CAT1/SLC7A1 binding domain using common B cell epitope fusion protein by pull down and neutralization assay.
|
