家蚕Ada3蛋白对杆状病毒复制及宿主细胞凋亡的影响  

Effect of Ada3 Protein in Bombyx mori on Baculovirus Replication and Host Cell Apoptosis

作　　者：朱亚杰 苗蒙 闫慧慧 全滟平[1,2] 于威 Zhu Yajie;Miao Meng;Yan Huihui;Quan Yanping;Yu Wei(College of Life Sciences and Medicine,Zhejiang Sci-Tech University,Hangzhou 310018,China;Zhejiang Key Labo-ratory of Silkworm Bioreactor and Biomedicine,Hangzhou 310018,China)

机构地区：[1]浙江理工大学生命科学与医药学院,杭州310018 [2]浙江省家蚕生物反应器和生物医药重点实验室,杭州310018

出　　处：《蚕业科学》2020年第2期171-177,共7页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金项目(31972623);浙江省自然科学基金项目(LY17C170006)。

摘　　要：基于BmNPV感染前后宿主细胞BmN的蛋白质差异组学研究结果,发现宿主细胞中的BmAda3(B.mori alteration/deficiency in activation 3)蛋白在BmNPV感染前后,其表达水平发生了显著变化。为探究BmAda3蛋白在病毒侵染过程中的作用,构建原核表达载体pET28a-ada3,通过诱导表达、纯化获得目的蛋白并制备多克隆抗体。将构建的瞬时表达载体pIEx-1-gfp-ada3转染BmN细胞,激光共聚焦显微镜观察发现BmAda3蛋白主要分布在细胞核中。将构建的瞬时表达载体pIEx-1-ada3转染BmN细胞,荧光定量PCR显示BmAda3蛋白的过表达可显著抑制病毒基因组复制。MTT细胞活力检测结果表明,BmAda3蛋白的过表达具有显著增强细胞活力的作用,DNA片段化和流式细胞术检测结果也进一步证实BmAda3蛋白的过表达可在一定程度上抑制BmNPV感染诱导的宿主细胞凋亡。研究结果将为深入了解家蚕细胞与BmNPV的互作调控关系提供一些新的线索。Based on our previous proteomics study on BmN cells before and after BmNPV infection,expression level of B.mori alteration/deficiency in activation 3(BmAda3)protein was found to change significantly.In order to further ex-plore the biological function of BmAda3 protein in the process of virus infection,prokaryotic expression vector pET28a-ada3 was constructed,and recombinant protein was obtained by induced expression and purified to prepare polyclonal antibody.After transient expression vector pIEx-1-gfp-ada3 was constructed to transfect BmN cells,observation under la-ser confocal microscope revealed that BmAda3 protein was mainly distributed in the nucleus.Then,transient expression vector pIEx-1-ada3 was constructed to transfect BmN cells.qPCR results showed that over-expression of BmAda3 protein could significantly inhibit replication of viral genome.In addition,MTT assay showed that over-expression of BmAda3 pro-tein enhanced cell viability significantly.DNA fragmentation and flow cytometry results further confirmed that over-expres-sion of BmAda3 protein could inhibit BmN cell apoptosis induced by BmNPV infection.This study will provide novel clues to further understand the interaction between silkworm host cell and BmNPV.

关 键 词：家蚕Ada3蛋白 BMNPV 病毒复制 细胞活力 细胞凋亡 

分 类 号：S881.24[农业科学—特种经济动物饲养] S884.5[农业科学—畜牧兽医]