Identification of novel receptor for bovine leukemia virus via cloning with common B cell epitope

通过常见 B 细胞表位克隆鉴定牛白血病病毒新型受体

• 批准号: 16F16404
• 负责人: 間 陽子
• 金额: $ 1.41万
• 依托单位: Institute of Physical and Chemical Research
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2016
• 资助国家: 日本
• 起止时间: 2016-10-07 至 2019-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16F16404/
• 关键词: Bovine leukemia virus; Receptor for BLV; CAT1/SLC7A1; Receptor binding domain; Large syncytia; CHO-K1 cells; Envelope glycoprotein (Env); bovine leukemia virus; CAT1/SLC7a1; BLV receptor; common B cell epitope; receptor binding domain; BLV; K

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.

CAT 1/SLC 7A 1已被鉴定为牛白血病病毒（BLV）的候选受体，但其是否为真正的受体尚不清楚。BLV与靶细胞上的细胞受体结合，其膜融合被激活，病毒进入宿主细胞。CHO-K1细胞不表达可检测的CAT 1/SLC 7A 1蛋白，并且对BLV感染具有抗性。因此，本研究从BLV天然宿主的CD 5 + B细胞cDNA中扩增了CAT 1/SLC 7A 1基因，并将其插入哺乳动物表达载体中。在与永久性BLV感染的FLK BLV细胞或FLK BLV细胞的培养上清液共培养的CAT 1/SLC 7A 1转染的CHO-K1细胞中检测到大的合胞体。这些结果清楚地表明，牛CAT 1/SLC 7A 1在抗性CHO-K1细胞中的表达赋予了对BLV感染的易感性。接下来，为了进一步确定CAT 1/SLC 7A 1是否充当BLV感染的细胞表面受体，我们使用流式细胞术验证了BLV颗粒与CAT 1/SLC 7A 1结合。结果表明，CAT 1/SLC 7A 1具有BLV附着受体的功能。第三，为了确定CAT 1/SLC 7A 1的结合结构域，扩增了共同的B细胞表位，并将其插入大肠杆菌表达载体pGEX中，将GST标签替换为His标签，表达并纯化融合蛋白。我尝试使用常见的B细胞表位融合蛋白通过下拉和中和试验来确定CAT 1/SLC 7A 1结合结构域。

项目成果

Development of diagnostic methods of BLV (in Japanese)

BLV诊断方法的开发（日语）

• 发表时间: 2017
• 期刊: Journal of Veterinary Medicine
• 作者: Lanlan Bai;Hirotaka Sato;Sonoko Watanuki;Shin-nosuke Takeshima;Yoko Aida.
• 通讯作者: Yoko Aida.

牛白血病ウイルス（BLV)抵抗性牛・感受性牛を利用したBLV清浄化対策の有効性

使用牛白血病病毒 (BLV) 抗性和易感牛进行 BLV 净化措施的有效性

• 发表时间: 2018
• 作者: 陸拾七;白らんらん;横山佳菜;広瀬智哉;佐藤洋隆;綿貫園子;米山洲二;小渕裕子;砂長伸司;藤田圭祐;田中なほ子;馬場未帆;竹嶋伸之輔;間陽子
• 通讯作者: 間陽子

A Vaccine Targeting Bovine Leukemia Virus Susceptible Cattle Suppresses Proviral Load

针对牛白血病病毒易感牛的疫苗可抑制原病毒载量

• 发表时间: 2017
• 作者: Yoko Aida;Lanlan Bai;Jiyun Kim;Pan He;Yuki Matsumoto;Noriaki Okimoto;Junya Yamagishi;Seiichi Tada;Yoshihiro Ito;Junko Kohara;Shin-nosuke Takeshima
• 通讯作者: Shin-nosuke Takeshima

乳汁中における牛白血病ウイルスの感染性評価とウシ主要組織適合との関連性

牛奶中牛白血病病毒感染性评价及其与牛主要组织相容性的关系

• 发表时间: 2018
• 作者: 綿貫園子;竹嶋伸之輔;佐藤洋隆;白らんらん;佐藤礼一郎;村上裕信;石崎宏;間陽子
• 通讯作者: 間陽子

牛白血病ウイルスのレセプター候補CAT1/SLC7a1の検証

牛白血病病毒候选受体CAT1/SLC7a1的验证

• 发表时间: 2018
• 作者: 白らんらん;佐藤洋隆;横山佳菜;間陽子
• 通讯作者: 間陽子