Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
Institute of Infectology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
J Gen Virol. 2011 Jun;92(Pt 6):1352-1357. doi: 10.1099/vir.0.029330-0. Epub 2011 Feb 23.
Bovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus within the family Flaviviridae. The lipid membrane of the virions is supposed to contain the three glycosylated envelope proteins E(rns), E1 and E2, but detailed studies of virus assembly are complicated because no efficient purification method for pestiviruses has been described so far. In this study, we generated infectious BVDV with N-terminally FLAG-tagged E(rns) or E2 proteins, respectively. The expression of the epitope-tagged E(rns) and E2 proteins could be shown by immunofluorescence and Western blot experiments. Furthermore, an affinity tag purification protocol for the isolation and concentration of infectious BVDV was established. In the preparation with a titre of 10(8.75) TCID(50) ml(-1), spherical particles with a diameter of 43-58 nm (mean diameter: 48 nm) could be detected by negative staining electron microscopy, and immunogold labelling located both E(rns) and E2 proteins at the virus membrane.
牛病毒性腹泻病毒（BVDV）属于黄病毒科瘟病毒属。病毒粒子的脂膜被认为包含三种糖基化的包膜蛋白 E(rns)、E1 和 E2，但由于迄今尚未描述有效的瘟病毒纯化方法，因此对病毒组装的详细研究变得复杂。在本研究中，我们分别生成了带有 N 端 FLAG 标签的 E(rns)或 E2 蛋白的感染性 BVDV。通过免疫荧光和 Western blot 实验可以证明表位标记的 E(rns)和 E2 蛋白的表达。此外，还建立了一种亲和标签纯化方案，用于分离和浓缩感染性 BVDV。在滴度为 10(8.75)TCID(50)ml(-1)的制备物中，通过负染色电子显微镜可以检测到直径为 43-58nm（平均直径：48nm）的球形颗粒，并且免疫金标记将 E(rns)和 E2 蛋白定位于病毒膜上。
