家蚕核型多角体病毒BmOrf99的研究  

Characterisation of BmOrf99 from Bombyx Mori Nucleopolyhedrovirus

作　　者：张曦[1] 胡小龙[1] 薛仁宇[1] 曹广力[1] 贡成良[1] 

机构地区：[1]苏州大学基础医学与生物科学学院,江苏苏州215123

出　　处：《安徽农业科学》2015年第21期43-50,共8页Journal of Anhui Agricultural Sciences

基　　金：国家重大基础研究计划"973"项目(2012CB114600)

摘　　要：[目的]探讨家蚕核型多角体病毒Bm Orf99在杆状病毒生命周期中的功能。[方法]运用Red同源重组技术构建Bm Orf99缺失型重组病毒,研究Bm Orf99对病毒复制、BV产生和病毒形态发生的影响。[结果]病毒转染细胞1.5 h可检测到Bm Orf99表达;通过Red重组系统构建敲除型病毒Bm NPV-Bm Orf99KO-polh-GFP,转染结果显示,敲除Bm Orf99病毒仍能增殖复制,透射电镜观察显示,Bm Orf99敲除对病毒粒子装配和多角体形成无明显影响。病毒DNA转染后24-72 h,敲除型病毒转染细胞培养上清BV水平略高于对照病毒Bm NPV-Bm Orf99WT-polh-GFP;转染后72 h,前者细胞培养上清TCID50略高于后者;转染6-72 h,敲除型病毒转染细胞中病毒DNA水平高于对照病毒转染细胞;敲除型病毒感染72 h后,细胞中Bm Orf98a表达水平比对照组提高2.21倍。[结论]Bm Orf99为病毒早期基因,且复制非必需,缺失可促进病毒DNA复制,略增加BV水平,但对病毒形态发生无影响,Bm Orf-99有可能通过调节病毒其他基因的表达而发挥作用。The aim was to the function of Bombyx rnori nucleopolyhedrovirus （BmNPV） BmOrt99 gene in the virus life cycle. [ Method ] To investigate the role of BmOrt99 gene during viral infection through the homologous recombination in Escherichia coll. [ Result ] The transcription of BmOrt99 was identified in the Bran cells after 1.5 h of transfeetion with baculovirus. BmOrf99-knockout virus （ BmNPV- BmOrO9KO-polh-GFP） was generated through the homologous recombination in Escherichia coll. Transfeetion results indicated that BmOrt99 was disappeared from the genome of BmNPV and had no effect on the virus proliferation. Electron microscopy exposed that occlusion derived virus can be produced in cells infected with the BmOrt99-knoekout virus. Further, virus DNA was transfeeted for the period of 24 to 72 hours and recorded that budded virion （BV） production and DNA replication in the BmN cells transfected with BmOrt99-knockout virus baemid were slightly higher than the cells transfeeted with wild type virus （BmNPV-BmOrt99WT-poIh-GFP）. Subsequent/y, TC1Dso of the BmOr199-knock- out virus bacmid was also slightly higher than wild type virus. The expression level of overlapping gene （BmOr198a） was enhanced by 2.21 fold after BmOrt99-knockout. [ Conclusion ] Outcome of the study revealed that BmOr/99 was an early gene and was not necessary for assembly of BmNPV, and absence of BmOr199 could be slightly increased the BV production and DNA replication. Our investigation helped to the researchers to unfold the function of BmOri99 gene interaction with other virus genes.

关 键 词：家蚕核型多角体病毒 BmOrf99 敲除 BV ODV 

分 类 号：S188[农业科学—农业基础科学]