重组杆状病毒AcMNPV-PK2-RFP诱导Sf9细胞凋亡的机制分析  

Apoptosis Mechanism Analysis of Sf9 Cells Infected by Recombinant Baculovirus AcMNPV-PK2-RFP

作　　者：卫丽丽 付月君[1] WEI Lili;FU Yuejun(Institute of Biotechnology,Shanxi University,Taiyuan 030006,China)

机构地区：[1]山西大学生物技术研究所,山西太原030006

出　　处：《山西农业科学》2019年第5期742-747,共6页Journal of Shanxi Agricultural Sciences

基　　金：国家自然科学基金项目(31272100);山西省自然科学基金面上项目(201801D121193)

摘　　要：苜蓿银纹夜蛾核型多角体病毒是一种模式杆状病毒,在农业虫害防治中广泛应用。其表达的PK2蛋白可以通过竞争性的与宿主细胞e IF2α激酶结合形成异源二聚体,抑制其磷酸化e IF2α,从而加速病毒增殖、诱导细胞凋亡。为了探讨PK2诱导宿主Sf9细胞凋亡的机制,利用Bac-to-Bac杆状病毒表达系统构建了重组杆状病毒AcMNPV-PK2-RFP。Western blot分析结果表明,重组病毒AcMNPV-PK2-RFP处理组在病毒侵染后48,60,72 h,宿主细胞内凋亡因子SfP53蛋白的表达量显著高于AcMNPV处理组,分别是AcMNPV处理组的1.16倍、1.39倍和1.79倍;在病毒侵染后48,72 h,AcMNPV-PK2-RFP处理组反映线粒体膜电位的绿色荧光分别是野生处理组的1.54倍和1.89倍,表明AcMNPV-PK2-RFP处理组的线粒体膜电位显著低于野生病毒处理组;同时,观察到AcMNPV-PK2-RFP处理组线粒体中的细胞色素c从病毒侵染后12 h开始释放。结果说明,过表达PK2的重组病毒AcMNPV-PK2-RFP可在感染后期加速SfP53蛋白的表达,通过影响线粒体凋亡信号通路加速宿主Sf9细胞的凋亡,同时表明重组病毒AcMNPV-PK2-RFP是一种相比野生型病毒抗虫活性更高的杀虫剂。Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most studied baculovirus species and is used widely in pest control. Ac-PK2 protein can increase viral fitness by engaging the N-lobe of the HRI-like insect eIF2α kinase domain and inhibiting the phosphorylation of eIF2α, which accelerates virus proliferation and induces host cell apoptosis. To investigate the mechanism of recombinant baculovirus AcMNPV-PK2-RFP induced apoptosis of host Sf9 cells, the recombinant baculovirus AcMNPV-PK2-RFP by Bac-to-Bac baculovirus expression system was constructed. Western blot analysis results showed that the expression of SfP53 protein in AcMNPV-PK2-RFP treatment group was significantly higher than that in the AcMNPV treatment group at 48, 60, 72 h after infection, and it was 1.16, 1.39, 1.79 times of that in AcMNPV treatment group, respectively. At 48, 72 h after infection, the green fluorescence of mitochondrial membrane potential in the AcMNPV-PK2-RFP treatment group was 1.54 times and 1.89 times of that in the AcMNPV treatment group, indicating that the mitochondrial membrane potential in AcMNPV-PK2-RFP treatment group was significantly lower than that in the wild virus treatment group. AcMNPV-PK2-RFP infection could promote the release of cytochrome c from mitochondria to cytoplasm from 12 h after infection was found. In summary, the recombinant virus AcMNPV-PK2-RFP accelerated expression of SfP53 protein at late stage of infection and induced the apoptosis of Sf9 cells by affecting the mitochondrial apoptosis signaling pathway, which suggested that AcMNPV-PK2-RFP virus had higher insecticidal activity than wild virus.

关 键 词：苜蓿银纹夜蛾核型多角体病毒(AcMNPV) Bac-to-Bac杆状病毒表达系统 SfP53蛋白 线粒体凋亡通路 草地贪夜蛾卵巢细胞Sf9 

分 类 号：S432.41[农业科学—植物病理学]