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2020, 41(2): 183-190. doi: 10.19663/j.issn2095-9869.20190709001

牡蛎疱疹病毒囊膜蛋白(ORF111)的基因克隆及表达

1. 大连海洋大学水产与生命学院 大连 116023;

2. 中国水产科学研究院黄海水产研究所 农业农村部海水养殖病害防治重点实验室 青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 青岛市海水养殖流行病学与生物安保重点实验室 青岛 266071

收稿日期:2019-07-09
修回日期:2019-07-31

基金项目:   农业农村部海水养殖病害防治重点实验室开放课题、中国水产科学研究院黄海水产研究所基本科研业务费(20603022019022)和现代农业产业技术体系(CARS-49)共同资助 

关键词: 牡蛎疱疹病毒 , 囊膜蛋白 , 原核表达 , 魁蚶

Gene Cloning and Expression of Ostreid Herpesvirus 1 Envelope Protein (ORF111)

1. Department of Fisheries and Life Science, Dalian Ocean University, Dalian 116023;

2. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology(Qingdao), Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Qingdao 266071

Received Date:2019-07-09
Accepted Date:2019-07-31

Keywords: Ostreid herpesvirus 1 , Envelope protein , Prokaryotic expression , Scapharca broughtonii

本研究对编码牡蛎疱疹病毒(Ostreid herpesvirus 1, OsHV-1)囊膜蛋白的orf111基因进行了生物信息学分析、克隆和表达。首先,通过特异性PCR扩增得到orf111基因全长序列,并对其编码囊膜蛋白的理化性质、高级结构、跨膜区和抗原决定簇等进行生物信息学分析。结果显示,orf111编码一种稳定的疏水性蛋白,具有5个跨膜结构域和9个抗原决定簇,同时,氨基酸序列中还包含1个高度保守的精氨酰-甘氨酰-天冬氨酸(Arg-Gly-Asp, RGD)结构域。随后,构建了pET28a-orf111重组质粒,并将其转化到大肠杆菌DH5α感受态细胞中。最后,通过使用异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达,表达产物分子量约为32 kDa。本研究应用原核表达得到了含RGD结构域的OsHV-1囊膜蛋白,为进一步制备ORF111蛋白单克隆抗体及开展牡蛎疱疹病毒侵染机制的研究奠定了重要基础,为将来OsHV-1的防控工作提供新的思路。

The envelope protein (ORF111) of Ostreid Herpesvirus 1 (OsHV-1) were studied using bioinformatics analysis, gene cloning and expression. Firstly, we designed the primers of the orf111 gene based on the complete genome sequence of OsHV-1, and successfully cloned the gene. We further analyzed the biological characteristics of the deduced protein sequence encoded by ORF111 gene, which including physicochemical properties, advanced structure, transmembrane region, and antigen determinant cluster. The results showed that the ORF111 protein is a stable hydrophobic protein, which contains nine antigenic determinants, five transmembrane domain structures, and a highly conservative pure ammonia arginyl-glycyl-aspartate (Arg-Gly-Asp, RGD) sequence. Then the OsHV-1-orf111 gene was linked with a pET28a(+) plasmid, and the recombinant plasmid pET28a-orf111 was successfully constructed. Finally, the recombinant plasmid was transformed into DH5α, and the recombinant protein was expressed after inducing by isopropyl-beta-D-thiogalactosine (IPTG). The SDS-PAGE test showed that the molecular weight of the ORF111 protein was about 32 kDa. In this study, an OsHV-1 envelope protein containing the RGD domain was successfully obtained by prokaryotic expression, which lays a foundation for the preparation of monoclonal antibodies of ORF111, and provides important basis for further study of the infection mechanism of OsHV-1. It also stimulates new ideas for the prevention and control of OsHV-1.

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牡蛎疱疹病毒囊膜蛋白(ORF111)的基因克隆及表达