摘要家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus, BmNPV)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BmNPV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BmN细胞，利用qRT-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。
Abstract：bro (baculovirus repeated ORF)-d gene of Bombyx mori nucleopolyhedrovirus (BmNPV) 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 BmN 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 qRT-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 BmNPV 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 virus-induced apoptosis of host cells, the bro-d-ko-Bacmid was co-transfected into BmN cells with the transient expression plasmid pIEx-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 pIEx-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 deficiency would strengthen the virus pathogenicity. Moreover, the B-cell lymphoma-2 (Bcl-2) protein and Bax (Bcl-2 assaciated X protein) expression levels in post-transfected cells and the key regulatory proteins in mitochondrial signaling death pathway were detected by Western blot. The results further confirmed that bro-d gene deletion would lead to significant decrease in the level of Bcl-2 protein (P＜0.05) and significant increase in the level of Bax protein (P＜0.05) compared with wild type virus, which resulted in a increased apoptotic level of host cells. These results indicated that bro-d gene could not only up-regulate the gene expression levels of different phages, but also could down-regulate the apoptotic level of host cells through mitochondrial pathway by regulating the expression of iap2. These results provide a new basis for understanding the function of bro-d gene in virus infection cycle, and a new way to transform the baculovirus expression system by prolonging the host cell life-span, and improve the expression amount of target proteins.
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