家蚕杆状病毒bro-d基因缺失对病毒基因转录和细胞凋亡的影响  

Effect of bro-d Deletion on the Viral Transcription and Cell Apoptosis in Silkworm(Bombyx mori)

作　　者：张媛媛[1] 魏铭[1] 李俊[1] 张婷[1] 全滟平[1] 舒特俊[1] 于威[1] 

机构地区：[1]浙江理工大学生命科学学院/浙江省家蚕生物反应器和生物医药重点实验室

出　　处：《农业生物技术学报》2016年第7期1073-1082,共10页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展计划(863)项目(No.2011AA100603);浙江理工大学521人才培养计划

摘　　要：家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,Bm NPV)bro(baculovirus repeated ORF)-d基因普遍存在于多种感染鳞翅目昆虫的多角体病毒基因组中,是杆状病毒中一类重复开放阅读框序列。为了解bro-d基因在病毒感染过程中的作用及其与宿主细胞凋亡之间的关系,本研究利用Red重组技术敲除Bm NPV基因组中的bro-d基因,构建基因缺失型病毒bro-d-ko-Bacmid,并将该缺失型病毒转染家蚕Bm N细胞,利用q RT-PCR技术检测bro-d基因的缺失对病毒基因组复制、转录水平以及抑制凋亡蛋白2基因(inhibitor of apoptosis protein 2,iap2)的影响。结果显示,bro-d基因缺失后会导致病毒基因组的复制水平显著下降(P<0.05),同时病毒的早期基因lef-3、晚期基因vp39和极晚期基因p10的转录水平也都显著下降(P<0.05);iap2的转录水平显著下调(P<0.05)。在bro-d基因缺失型病毒的复制水平明显低于野生型病毒的情况下,仍会导致细胞活力显著下降(P<0.05);bro-d基因缺失引起细胞B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)蛋白含量明显低于野生型病毒,而Bcl-2相关X蛋白(Bcl-2 assaciated X protein,Bax)含量显著高于野生型病毒(P<0.05),进而导致细胞凋亡水平上升。结果表明,bro-d基因不仅对病毒各时期基因表达水平具有调控作用,也可通过调控iap2的表达进而调控宿主细胞的凋亡水平。研究成果为深入了解bro-d基因在病毒感染过程中的功能提供了基础资料,也为通过延长宿主细胞寿命、提高目的蛋白表达产量来进行杆状病毒表达载体的改造提供了新思路。bro（baculovirus repeated ORF）-d gene of Bombyx mori nucleopolyhedrovirus（Bm NPV） is a kind of repeated open reading frame sequence in baculovirus and is widespread in a variety polyhedrosis virus genome which specially infects Lepidopteran insect. In order to understand the role of bro- d gene in viral infection and the relationship with host cell apoptosis, the bro-d gene was knocked out by Red recombination system to construct the bro-d-deficiency type virus（bro-d-ko-Bacmid）, and was encapsulated by liposome and then was transfected into Bm N cells, and wild type bacmid（Wt-Bacmid） was used as the control. The effects of bro-d gene on the replication and transcription level of virus genome and inhibition of apoptosis protein 2（iap2） were detected by q RT-PCR, and the results showed that bro-d gene deletion would lead to a significant decrease on the replication level of viral genome and the transcription levels of viral early gene lef-3, late gene vp39 and very late gene p10 would be also significant decrease（P〈0.05）. These results indicated that bro-d gene was a nonessential gene for Bm NPV replication and it was involved in the regulatory process of viral gene in different transcription phase. In order to further explore the effects of bro- d gene deletion on virusinduced apoptosis of host cells, the bro- d- ko- Bacmid was co- transfected into Bm N cells with the transient expression plasmid p IEx- 1- bro- d which contained the IE promoter which only triggered in eukaryon. The results showed that the transcription level of iap2 was decreased significantly（P〈0.05） in the cells transfected with bro- d- ko- Bacmid and was increased after cotransfected with p IEx- 1- bro- d. Furthermore, in spite of the viral replication and transcription levels of bro-d-ko-Bacmid were significantly lower than that of Wt- Bacmid（P〈0.05）, and bro- d- ko- Bacmid would cause a significant decrease in cell viability compared with Wt- Bacmid（P〈0.05）. These results implied that bro- d deficien

关 键 词：家蚕核型多角体病毒(BmNPV) bro-d 基因敲除 转录 细胞凋亡 

分 类 号：Q523.3[生物学—生物化学]