Bruce L D, Trumper B B, Lightner D V
Department of Veterinary Science, University of Arizona, Tucson 85721.
J Virol Methods. 1991 Oct;34(3):245-54. doi: 10.1016/0166-0934(91)90104-8.
Procedures for the purification of virions and nucleocapsids of Baculovirus penaei (BP) of penaeid shrimp and subsequent extraction of the viral nucleic acid are described. BP-infected hepatopancrata, from two species of shrimp from different geographical locations in the Americas, were removed and homogenized in a solution of TN buffer (0.01 M Tris-HCl, 0.10 M NaCl, pH 8.0). The homogenized mixture was strained through a 100-mesh screen to remove large pieces of tissue and centrifuged to concentrate the remaining material. The pellet was suspended in TN buffer and layered on to a handmade CsCl gradient. Fractions were collected according to the bands observed in the gradient, and the optical density at 254 nm was recorded for each fraction. The resultant data was tabulated and graphed. Additionally, each fraction was examined by transmission electron microscopy to determine relative numbers of viral particles present. Large amounts of virus consistently corresponded to a specific band in the gradient, which produced a peak when the spectrophometric data was graphed. Nucleic acid was then extracted from the purified viral particles. Removal of polysaccharides was accomplished with the addition of CTAB/NaCl. The BP DNA was visualized on an agarose gel with phage lambda DNA markers for size estimation, and a preliminary endonuclease digestion was performed using BamHI.
本文描述了对来自对虾的杆状病毒对虾（BP）的病毒粒子和核衣壳进行纯化以及随后提取病毒核酸的程序。从美洲不同地理位置的两种虾中取出受BP感染的肝胰腺，在TN缓冲液（0.01 M Tris-HCl，0.10 M NaCl，pH 8.0）溶液中匀浆。将匀浆混合物通过100目筛网过滤以去除大块组织，然后离心以浓缩剩余物质。将沉淀悬浮在TN缓冲液中，并铺在手工制作的CsCl梯度上。根据梯度中观察到的条带收集级分，并记录每个级分在254 nm处的光密度。将所得数据制成表格并绘图。此外，通过透射电子显微镜检查每个级分，以确定存在的病毒颗粒的相对数量。大量病毒始终对应于梯度中的一个特定条带，当绘制分光光度数据时，该条带会产生一个峰值。然后从纯化的病毒颗粒中提取核酸。通过添加CTAB/NaCl去除多糖。使用噬菌体λ DNA标记物在琼脂糖凝胶上观察BP DNA以进行大小估计，并使用BamHI进行初步的核酸内切酶消化。
