家蚕核型多角体病毒极晚期基因polh和p 10启动子结合蛋白的分析鉴定

Identification of the Proteins Binding with the Promoters of Very Late Genes polh and p 10 in Bombyx mori Nucleopolyhedrovirus(BmNPV)

核型多角体病毒的多角体蛋白基因polh和p 10基因是杆状病毒极晚期转录表达的重要基因,具有超高水平表达的显著特征。通过DNA pull-down技术筛选家蚕核型多角体病毒(BmNPV)polh和p 10基因启动子序列上的结合蛋白,再利用液相色谱-串联质谱(LC-MS/MS)技术分析鉴定与2个基因启动子结合的宿主和病毒蛋白,旨在从转录水平上更深入地揭示影响极晚期基因转录启动的调节因子。结果表明,与2个极晚期基因启动子共同结合的蛋白质有47个,其中10个是病毒自身编码的蛋白质,另外37个是宿主蛋白。进一步分析表明,这10个病毒蛋白大多具有结合DNA的属性,包括病毒自身编码的RNA聚合酶亚基;37个宿主蛋白中有细胞骨架蛋白,其中肌动蛋白(actin)肽段覆盖率很高。对polh和p 10基因启动子结合蛋白的分析鉴定将有助于在转录水平上深入阐明杆状病毒极晚期基因超高水平表达的分子机制,同时也可为提高杆状病毒表达系统的表达效率提供理论参考。

Baculovirus genes polh and p 10 were hyperexpressed at very late stages in viral life cycle.In this study,the proteins binding with the polh and p 10 promoters were screened by DNA pull-down and analyzed by liquid chromatography-tandem mass spectrometry(LC-MS/MS),aiming to reveal the transcriptional regulators that affect the transcription initiation of very late genes at the transcriptional level.The results showed that 47 binding proteins of polh and p 10 promoters were identified,of which 37 binding proteins were host-encoded proteins and 10 binding proteins were viral proteins.Furthermore,most of the 10 viral proteins were found to be DNA-binding proteins,including the RNA polymerase subunits encoded by the virus itself.Among the 37 host proteins,there were cytoskeletal proteins,among which actin had a high coverage rate.The above results were bene-ficial to further understand the molecular mechanism of hyperexpression of very late genes in baculovirus at the transcriptional level,and also provided a theoretical guidance for improving the expression efficiency of baculo-virus expression system.