DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines
DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines
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DOI：
10.1007/s00705-002-0922-3
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发表时间：
2003-03
影响因子：
2.7
通讯作者：
Junya Yamagishi;R. Isobe;T. Takebuchi;H. Bando
中科院分区：
文献类型：
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作者：
Junya Yamagishi;R. Isobe;T. Takebuchi;H. Bando
我们描述，第一次，病毒DNA芯片的产生，同时表达测量几乎所有已知的开放阅读框架（ORF）的最好的研究成员的家庭杆状病毒科，苜蓿银纹夜蛾多核型多角体病毒（AcMNPV）和家蚕核型多角体病毒（BmNPV）。在这项研究中，一个病毒的DNA芯片（Ac-BmNPV芯片）的制作和用于表征病毒基因表达谱AcMNPV在不同类型的细胞。病毒芯片由病毒DNA的微阵列组成，所述病毒DNA的微阵列通过将PCR扩增的病毒DNA片段机器人沉积在玻璃上以用于NPV基因组中的ORF。用荧光标记的cDNA与DNA芯片杂交，检测病毒基因的表达，所述cDNA是从感染的草地贪夜蛾（Spodoptera frugiperda）、Sf 9细胞和粉纹夜蛾（Trichoplusia ni）、TnHigh-Five细胞制备的，后者是杆状病毒和重组蛋白的主要生产者。比较AcMNPV已知ORF的表达谱，阐明了6个基因（ORF 150、p10、pk 2和3个晚期基因表达因子基因lef-3、p35和lef-6），其中每一个基因的表达在两个细胞系中受到不同的调控。已知这些基因中的大多数与病毒的生命周期密切相关，如DNA复制、晚期基因表达和感染细胞中多角体的释放。这些结果意味着这些病毒基因的差异表达解释了这两种细胞系之间病毒复制的差异。因此，这些制作的NPV DNA微阵列，允许在病毒基因组水平上的基因表达的快速分析，应该大大加快了大基因组的NPV的功能分析。
We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the familyBaculoviridae,Autographa californica multiple nucleopolyhedrovirus(AcMNPV) andBombyx mori nucleopolyhedrovirus(BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infectedSpodoptera frugiperda, Sf9 cells andTrichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150,p10,pk2, and three late gene expression factor geneslef-3,p35andlef-6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV.