Müller R, Pearson M N, Russell R L, Rohrmann G F
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331.
Virology. 1990 May;176(1):133-44. doi: 10.1016/0042-6822(90)90238-m.
Two lambda gt11 clones containing overlapping DNA inserts encoding portions of a structural protein gene from Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) were identified by their immunoreactivity with polyclonal antisera produced against purified polyhedra-derived virus. Sequence analysis of a 3.6-kb region of the baculovirus genome (map units 69.1-71.6) from which the lambda gt11 inserts originated revealed an open reading frame of 1872 nt (624 amino acids) encoding a predicted protein of 70.6 kDa. Northern blot, primer extension, and 3' S1 analysis of this ORF indicated that an mRNA of approximately 2100 nt was transcribed from this gene. The mRNA appears to initiate from a late promoter/mRNA start site consensus sequence GTAAG and is expressed at late times postinfection. A gene fusion containing the C-terminal 368 amino acids of the gene was constructed using a bacterial trpE expression vector. Rabbit antiserum made against the purified fusion protein reacted with a protein of 87 kDa on Western blots of infected cell extracts at 24 hr p.i. and thereafter. The p87 protein was shown to be a component of both budded and polyhedra-derived virus and purified capsids. Immunofluorescence analysis indicated that p87 is expressed late in infection and concentrated in infected cell nuclei.
通过与针对纯化的多角体来源病毒产生的多克隆抗血清的免疫反应性，鉴定出两个λgt11克隆，它们包含编码来自云杉芽蛾多粒包埋核型多角体病毒（OpMNPV）结构蛋白基因部分的重叠DNA插入片段。对杆状病毒基因组中λgt11插入片段所源自的3.6 kb区域（图谱单位69.1 - 71.6）进行序列分析，发现一个1872 nt（624个氨基酸）的开放阅读框，编码一个预测分子量为70.6 kDa的蛋白质。对该开放阅读框进行Northern印迹、引物延伸和3'S1分析表明，该基因转录出一个约2100 nt的mRNA。该mRNA似乎从晚期启动子/mRNA起始位点共有序列GTAAG起始转录，并在感染后期表达。使用细菌trpE表达载体构建了一个包含该基因C末端368个氨基酸的基因融合体。针对纯化融合蛋白制备的兔抗血清在感染后24小时及之后的感染细胞提取物的Western印迹中与一个87 kDa的蛋白质发生反应。p87蛋白被证明是出芽病毒和多角体来源病毒以及纯化衣壳的组成成分。免疫荧光分析表明，p87在感染后期表达，并集中在感染细胞核中。
