Gombart A F, Pearson M N, Rohrmann G F, Beaudreau G S
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331.
Virology. 1989 Mar;169(1):182-93. doi: 10.1016/0042-6822(89)90054-8.
Using a polyclonal mouse antiserum produced against purified virions of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV), two immunoreactive lambda gtII clones were identified which contained nonoverlapping insert DNAs which mapped to a single open reading frame (ORF) in the HindIII-M fragment. Analysis of nucleotide sequence data indicates that this ORF encodes a protein with a MW of 32.4 kDa. A trpE-p32 gene fusion containing the entire p32 ORF was constructed, and the fusion protein was purified and used to immunize rabbits. Western blot analysis and immunofluorescence studies using the anti-TrpE-p32 antiserum detected a polyhedra-derived virus (PDV)-associated protein of 32 kDa at 24 hr postinfection (hr p.i.). The protein was observed in the cytoplasm and nucleus at 24 hr p.i. and became concentrated in the cytoplasm late in infection. Western blot analysis and immunofluorescent microscopy of polyhedra solubilized under various conditions indicated that p32 is associated with the polyhedral envelope. The predicted amino acid sequence for p32 showed 58% amino acid identity with the predicted amino acid sequence for an ORF (ORF 3) in a similar region of the genome of the MNPV of Autographa californica (AcMNPV). The solubility properties of the p32 protein and reciprocal immunoblotting experiments indicate the OpMNPV p32 gene encodes a protein which is homologous to the polyhedral envelope-associated phosphoprotein of AcMNPV, pp34, recently reported by M.A. Whitt and J.S. Manning [(1988) Virology 163, 33-42].
利用针对云杉芽卷蛾多核衣壳核型多角体病毒（OpMNPV）纯化病毒粒子制备的多克隆小鼠抗血清，鉴定出两个免疫反应性λgtII克隆，它们含有不重叠的插入DNA，这些DNA定位于HindIII - M片段中的单个开放阅读框（ORF）。核苷酸序列数据分析表明，该ORF编码一种分子量为32.4 kDa的蛋白质。构建了包含整个p32 ORF的trpE - p32基因融合体，纯化融合蛋白并用其免疫兔子。使用抗TrpE - p32抗血清进行的蛋白质免疫印迹分析和免疫荧光研究在感染后24小时（h p.i.）检测到一种32 kDa的多面体衍生病毒（PDV）相关蛋白。该蛋白在感染后24小时在细胞质和细胞核中可见，并在感染后期集中在细胞质中。对在各种条件下溶解的多角体进行的蛋白质免疫印迹分析和免疫荧光显微镜检查表明，p32与多面体包膜相关。p32的预测氨基酸序列与苜蓿银纹夜蛾核型多角体病毒（AcMNPV）基因组相似区域中的一个ORF（ORF 3）的预测氨基酸序列显示出58%的氨基酸同一性。p32蛋白的溶解性特性和相互免疫印迹实验表明，OpMNPV p32基因编码一种与AcMNPV的多面体包膜相关磷蛋白pp34同源的蛋白质，pp34最近由M.A.惠特和J.S.曼宁报道[（1988年）《病毒学》163，33 - 42]。
