Identification of bluetongue virus VP6 protein as a nucleic acid-binding protein and the localization of VP6 in virus-infected vertebrate cells.
Identification of bluetongue virus VP6 protein as a nucleic acid-binding protein and the localization of VP6 in virus-infected vertebrate cells.
复制标题
蓝舌病毒 VP6 蛋白作为核酸结合蛋白的鉴定以及 VP6 在病毒感染的脊椎动物细胞中的定位。
DOI：
10.1128/jvi.64.1.1-8.1990
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发表时间：
1990
影响因子：
5.4
通讯作者：
Thomas,CP
中科院分区：
文献类型：
--
作者：
Roy,P;Adachi,A;Urakawa,T;Booth,TF;Thomas,CP
最近，昆虫杆状病毒苜蓿银纹夜蛾核多角体病毒（AcNPV）已被有效地改造为昆虫细胞中表达各种基因的高效载体。编码小核心蛋白 (VP6) 的蓝舌病毒血清型 10（BTV-10，呼肠孤病毒科环状病毒成员）的 RNA 片段 9 的 cDNA 序列已插入源自 AcNPV 的 pAcYM1 转移载体的 BamHI 位点。在真实的 AcNPV DNA 存在下，将衍生载体与草地贪夜蛾细胞共转染，以产生重组病毒。它们合成了大量的蛋白质（约占染色细胞蛋白质的 50%），其大小和抗原性与真正的 BTV VP6 相似。通过使用 RNA 覆盖蛋白印迹测定，将表达的蛋白鉴定为核酸结合蛋白。使用表达的 VP6 蛋白制备的多克隆抗 VP6 血清已用于免疫金程序，以在 BTV 感染的哺乳动物细胞中定位 VP6。人们发现金与病毒包涵体 (VIB) 基质、VIB 中的病毒样颗粒以及靠近 VIB 或从细胞释放并吸附到细胞表面的成熟病毒颗粒有关。重组病毒抗原也已用于鉴定受感染绵羊血清中不同 BTV 血清型的抗体，表明表达的蛋白作为群体反应抗原用于诊断 BTV 感染的潜力。
Recently the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has been effectively adapted as a highly efficient vector in insect cells for the expression of various genes. A cDNA sequence of RNA segment 9 of bluetongue virus serotype 10 (BTV-10, an orbivirus member of the Reoviridae family) encoding a minor core protein (VP6) has been inserted into the BamHI site of the pAcYM1 transfer vector derived from AcNPV. Spodoptera frugiperda cells were cotransfected with the derived vector in the presence of authentic AcNPV DNA to produce recombinant viruses. These synthesized significant amounts of a protein (representing ca. 50% of the stained cellular protein) similar in size and antigenicity to the authentic BTV VP6. The expressed protein was identified as a nucleic acid-binding protein by using an RNA overlay-protein blot assay. A polyclonal anti-VP6 serum prepared by using the expressed VP6 protein has been used in an immunogold procedure to locate VP6 in BTV-infected mammalian cells. Gold was found to be associated with the matrix of virus inclusion bodies (VIB), with viruslike particles in the VIB, as well as with mature virion particles that were in close proximity to the VIB or were released from cells and adsorbed to cell surfaces. The recombinant virus antigen has also been used to identify antibodies to different BTV serotypes in infected sheep sera, indicating the potential of the expressed protein as a group-reactive antigen for the diagnosis of BTV infections.