牡蛎疱疹病毒结构蛋白真核表达系统构建及多聚化特性  

Construction of and polymerizing characterization with eukaryotic expression system for Ostreid herpesvirus 1 structural proteins

作　　者：曹书华 魏茂乐 李永仁 黄博闻 辛鲁生 白昌明[2] 王崇明[2] CAO Shuhua;WEI Maole;LI Yongren;HUANG Bowen;XIN Lusheng;BAI Changming;WANG Chongming(Tianjin Key Laboratory of Aqua-Ecology and Aquaculture,College of Fishery,Tianjin Agriculture University,Tianjin 300384,China;State Key Laboratory of Mariculture Biobreeding and Sustainable Goods,Laboratory for Marine Fisheries Science and Food Production Processes,Key Laboratory of Maricultural Organism Disease Control,Ministry of Agriculture and Rural Affairs,Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071,China)

机构地区：[1]天津农学院水产学院,天津市水产生态及养殖重点实验室,天津300384 [2]中国水产科学研究院黄海水产研究所,海水养殖生物育种与可持续产出全国重点实验室,海洋渔业科学与食物产出过程功能实验室,农业农村部海水养殖病害防治重点实验室,青岛市海水养殖流行病学与生物安保重点实验室,山东青岛266071

出　　处：《水产学报》2024年第5期63-72,共10页Journal of Fisheries of China

基　　金：国家自然科学基金(32073014);中国水产科学研究院基本科研业务费专项(2023TD30);黄海水产研究所基本科研业务费专项(20603022023010);国家现代农业产业技术体系(CARS-49);山东省博士后创新项目(SDCX-ZG-202303048)。

摘　　要：牡蛎疱疹病毒(OsHV-1)在全球范围内导致牡蛎、扇贝与蚶类的大规模死亡,成为双壳贝类养殖产业的重要威胁。为了解OsHV-1的结构与致病机制。本研究利用人胚胎肾细胞(HEK293t),构建OsHV-1主要核衣壳蛋白(ORF104和ORF33)的真核表达系统,并对ORF104和ORF33潜在相互作用进行分析。实验首先通过特异性PCR扩增技术得到orf 104和orf 33的基因序列,根据其编码蛋白的理化性质、跨膜区与三维结构等生物信息学分析结果,选择pCDNA3.1(+)构建两种基因的重组表达质粒。重组质粒经大肠杆菌扩增、提取后,利用转染试剂Lipo8000™将pCDNA3.1(+)-orf 104与pCDNA3.1(+)-orf 33分别单独或共转染至HEK293t。然后,将转染后的细胞培养18 h后裂解收集蛋白。最后利用蛋白免疫印迹(Western blot,WB)与负染电镜检测两种目的蛋白的表达情况。结果显示,实验成功构建了OsHV-1衣壳蛋白ORF104和ORF33的重组表达质粒载体,通过真核细胞表达得到大小约为135与35 ku的目的蛋白。研究表明,表达质粒可在真核表达系统中实现蛋白单独转染与共转染,共转染蛋白间可能存在相互作用的趋势并形成多聚体,其中,ORF33自身即可形成分子质量不同的多聚体。本研究首次利用真核表达系统开展OsHV-1关键结构蛋白的表达,为进一步开展该病毒结构蛋白功能与互作,以及病毒入侵机制研究奠定基础。Ostreid herpesvirus 1(OsHV-1)infection has been linked to mass mortality of cultivated mollusks of different species worldwide.In this study,we established a eukaryotic system for the expression of ORF104 and ORF33 of OsHV-1,and investigated the polymerizing characterization of ORF104 and ORF33.Firstly,the biochemical characteristics,typical domains and 3D structures were analyzed bioinformatically.Secondly,we constructed recombinant plasmids for the two genes,and individually transfected or co-transfected them into Human Embryonic Kidney Cells(HEK293t).Finally,the expression and polymerization of the two target proteins were investigated using Western blot(WB)and electron microscopy.Bioinformatic analysis demonstrated that hydrophilicity of ORF33 and ORF104 were−0.49 and 0.82,making them stable hydrophilic and hydrophobic proteins respectively.For the construction of recombinant plasmids,genes of ORF33 and ORF104 were amplified with specifically designed primers,which generated PCR products of about 1000 bp and 3500 bp for ORF33 and ORF104 respectively.The recombinant plasmids were transmitted into HEK293t cells using Lipo8000TM for expression.The target bands around 140 ku and 35 ku in length were obtained by WB analysis after SDS-PAGE.However,the interaction between the two viral proteins was still undetermined.The ORF104 protein was identified by electron microscopy,which had a diameter of approximately 20 nm.Due to the small diameter of ORF33,it could not be clearly resolved with the background particles under negative staining electron microscopy in the present study.When the products of the two co-transfected plasmids were investigated,the diameter of the particles was between 10 and 30 nm.In summary,we established a method for the expression of OsHV-1 structural proteins using the eukaryotic expression system for the first time.Polymerization of the two proteins individually and together was investigated and characterized.The method described here provides a new approach for further research on the func

关 键 词：牡蛎疱疹病毒 核衣壳蛋白 真核表达系统 蛋白多聚化 

分 类 号：S944.4[农业科学—水产养殖]