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.

CAT1/SLC7A1已被确定为牛白血病病毒（BLV）的候选受体，但是它是否是实际受体仍然未知。 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细胞未表达可检测到的CAT1/SLC7A1蛋白，并且对BLV感染具有抗性。因此，我从BLV天然宿主的CD5+ B细胞的cDNA扩增了CAT1/SLC7A1基因，并插入了哺乳动物表达载体中。在CAT1/SLC7A1转染的CHO-K1细胞中检测到大型合胞菌，这些CHO-K1细胞与永久感染BLV的FLK-BLV细胞或FLK-BLV细胞的培养上清液共培养。这些结果清楚地表明，牛CAT1/SLC7A1在抗性CHO-K1细胞中表达易受到BLV感染的影响。接下来，为了进一步确定CAT1/SLC7A1是否充当BLV感染的细胞表面受体，我们验证了使用流式细胞仪与CAT1/SLC7A1结合的BLV颗粒。结果表明，CAT1/SLC7A1充当BLV附着受体。第三，为了确定CAT1/SLC7A1结合结构域，将常见的B细胞表位放大并插入大肠杆菌表达载体PGEX中，该vSTAG被用His-TAG代替以表达和纯化融合蛋白。我试图通过下和中和测定法使用常见的B细胞表位融合蛋白来确定CAT1/SLC7A1结合域。

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.

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

蛍光発現レポーター細胞を用いた牛白血病ウイルスのレセプター候補CAT1/SLC7a1の検証

使用荧光报告细胞验证牛白血病病毒候选受体CAT1/SLC7a1

• 发表时间: 2018
• 作者: Bai Lanlan;佐藤洋隆;横山佳菜;間陽子
• 通讯作者: 間陽子

白血病ウイルスの共通B細胞エピトープとその抗体結合部位の同定

白血病病毒常见B细胞表位及其抗体结合位点的鉴定

• 发表时间: 2016
• 作者: Bai Lanlan;竹嶋伸之輔;磯貝恵美子;間陽子
• 通讯作者: 間陽子

牛白血病感受性における牛白血病ウイルス様粒子ワクチンによるプロウイルス量の上昇抑制効果

牛白血病病毒样颗粒疫苗抑制原病毒水平升高对牛白血病易感性的影响

• 发表时间: 2017
• 作者: 間陽子;大附寛幸;Lanlan Bai;根岸冴木;綿貫園子;佐藤洋隆;竹嶋伸之輔
• 通讯作者: 竹嶋伸之輔