杆状病毒核心基因ac78的缺失影响多粒包埋型病毒粒子形成的分子机制研究

项目名称： 杆状病毒核心基因ac78的缺失影响多粒包埋型病毒粒子形成的分子机制研究

项目编号： No.31501705

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 农业科学

项目作者： 李赛男

作者单位： 肇庆学院

项目金额： 18万元

中文摘要： 苜蓿丫纹夜蛾核多角体病毒（AcMNPV）ac78是最近被发现的杆状病毒核心基因，缺失该基因的病毒在感染的细胞中多粒包埋型病毒（M-ODV）的产生显著减少。生物信息学分析显示Ac78可能具有细胞色素C氧化酶活性。本项目拟以ac78为研究对象，应用分子生物学、免疫组织化学、免疫沉淀等技术和手段，原核表达并纯化Ac78，检测Ac78的酶活性；在野生型和ac78缺失型bacmid中分别构建p33补回型重组病毒，利用这些重组病毒分析Ac78与杆状病毒巯基氧化酶P33的关系；利用免疫沉淀和免疫共沉淀技术，确定Ac78和P33通过与病毒中的哪些蛋白相互作用来行使功能，探讨AcMNPV中与M-ODV形成相关的可能的二硫键形成还原通路。深入研究Ac78参与M-ODV形成的分子机制对揭示杆状病毒的复制与侵染机制具有重要意义，也为更好地利用杆状病毒防治害虫和构建遗传改良基因工程杆状病毒提供重要的理论依据。

中文关键词： 杆状病毒；ac78；多粒包埋型病毒粒子；分子机制；还原通路

英文摘要： Baculoviruses are a very diverse group of insect-specific viruses with rod-shaped, enveloped, double-stranded DNA genomes that specifically infect insect species mainly from the orders Lepidoptera, Hymenoptera, and Diptera. ac78 of the archetype of the Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is a recently identified baculovirus core gene. Our previous studies showed that the deletion of ac78 severely affected the morphogenesis of multiple nucleocapsid-enveloped occlusion-derived virions (M-ODVs). Bioinformatics analysis showed that Ac78 contains a putative cytochrome C oxidase N-terminal region, which has been shown to be important in the M-ODV morphogenesis. Here, we hypothesize that Ac78 may have cytochrome C oxidase activity; Ac78may work together with the baculovirus sulfhydryl oxidase P33 in a redox pathway for protein disulfide bond formation, which may be related to the formation of M-ODVs..In this study, we will be working on the role of ac78 in the formation of M-ODVs using various molecular biology, immunohistochemistry, immunoprecipitation techniques and methods. To investigate whether Ac78 has cytochrome C oxidase activity, Ac78 will be prokaryotic expressed in Escherichia coli and purified, and the cytochrome C oxidase activity of Ac78 will be assayed using a Purified Cytochrome C Oxidase Activity Assay Kit. To detect the interrelation of Ac78 with baculovirus sulfhydryl oxidase P33, two p33 repaired viruses will be constructed, in which the p33 gene with its native promoter and ORF plus a FLAG tag fused in-frame to the C terminus of p33 ORF is inserted into the polh locus of ac78 deletion bacmid bAc78KO and AcMNPV bacmid bMON14272, respectively. Sf9 cells will be transfected with these recombinant viruses and be collected at the designated times post transfection. The sulfhydryl oxidase activity of P33 in Sf9 cells will be detected, and the reductive degree of P33 sulfhydryl will be analyzed by Western blotting. Using immunoprecipitation and co-immunoprecipitation, we will investigate the baculovirus proteins which have interaction with Ac78 or P33, respectively. Subsequently, these protein repaired viruses will be constructed on the basis of p33 deletion bacmid bAc92KO, bAc78KO and bMON14272, respectively. To investigate whether AcMNPV has a redox pathway for protein disulfide bond formation which is related to the formation of M-ODVs, these recombinant viruses will be used to study the redox relationship of these proteins with Ac78 or P33..In summary, this study will further investigate the molecular mechanism for the effects of deletion of ac78 on the the formation of M-ODVs. This research will be important for revealing the replication and infection mechanism of baculovirus. It also will provide important theoretical basis for the better use of baculovirus to control pests and construct improved genetic engineering baculovirus.

英文关键词： baculovirus;ac78;multiple nucleocapsid-enveloped occlusion-derived virion;molecular mechanism;redox pathway