EXPRESSION, CHARACTERIZATION, AND PURIFICATION OF A PHOSPHORYLATED RABIES NUCLEOPROTEIN SYNTHESIZED IN INSECT CELLS BY BACULOVIRUS VECTORS
EXPRESSION, CHARACTERIZATION, AND PURIFICATION OF A PHOSPHORYLATED RABIES NUCLEOPROTEIN SYNTHESIZED IN INSECT CELLS BY BACULOVIRUS VECTORS
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DOI：
10.1016/0042-6822(90)90346-s
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发表时间：
1990-10-01
期刊：
影响因子：
3.7
通讯作者：
BISHOP, DHL
中科院分区：
文献类型：
--
作者：
PREHAUD, C;HARRIS, RD;BISHOP, DHL
构建了狂犬病病毒核型多角体病毒(AcNPV)杆状病毒表达载体AcNPV3，该载体含有狂犬病病毒N基因的完整编码区。该基因被置于AcNPV多角体启动子的控制下，并得到高水平的表达(66 mg N蛋白/升2倍)。109个细胞)，用衍生的重组病毒利用果翅夜蛾细胞系。利用已有的抗血清，通过与狂犬病病毒天然N蛋白的比较，证实N蛋白的抗原性与狂犬病病毒天然N蛋白相似。对表达蛋白的鉴定表明，像哺乳动物细胞培养的CVS病毒的N蛋白一样，N蛋白是磷酸化的。表达的狂犬病N蛋白诱导小鼠产生抗体，该抗体与狂犬病病毒蛋白发生强烈反应。通过差速离心和离子交换层析，从昆虫细胞中回收了表达的核蛋白。表达的狂犬病N蛋白是适合病毒诊断和结构研究的真实蛋白来源。
A baculovirus expression vector (AcNPV3) derived from the nuclear polyhedrosis virus of Autographa californica (AcNPV) was prepared containing the complete coding region of the nucleoprotein (N) gene of rabies virus (Gif-sur-Yvette clone of the CVS strain). The gene was placed under the control of the AcNPv polyhedrin promoter and was expressed to high levels (66 mg N protein/liter of 2 .times. 109 cells) by the derived recombinant virus using a Spodoptera frugiperda cell line. Using available antisera, it was established that the antigenic characteristics of the N protein were similar by comparison with those of the native N protein of rabies virus. Characterization of the expressed protein established that, like the N protein of mammalian cell-grown-CVS virus, the N protein was phosphorylated. The expressed rabies N protein induced antibodies in mice that reacted strongly with the rabies viral protein. The expressed nucleoprotein was recovered from the insect cells by differential centrifugation followed by ion exchange chromatography. The expressed rabies N protein represents a source of authentic protein suitable for virus diagnosis as well as structural studies.