| Ⅱ型鲤疱疹病毒ORF121蛋白的多克隆抗体制备及鉴定 |
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| 引用本文： | 余琳,吕利群,王浩. Ⅱ型鲤疱疹病毒ORF121蛋白的多克隆抗体制备及鉴定[J]. 水产学报, 2019, 43(6): 1463-1471 |
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| 作者姓名： | 余琳 吕利群 王浩 |
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| 作者单位： | 上海海洋大学, 国家水生动物病原库, 上海 201306,上海海洋大学, 国家水生动物病原库, 上海 201306;上海海洋大学, 农业农村部淡水种质资源重点实验室, 上海 201306;上海海洋大学, 水产科学国家级实验教学示范中心, 上海 201306,上海海洋大学, 国家水生动物病原库, 上海 201306;上海海洋大学, 农业农村部淡水种质资源重点实验室, 上海 201306;上海海洋大学, 水产科学国家级实验教学示范中心, 上海 201306 |
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| 基金项目： | 第四届中国科协青年人才托举工程（中国水产学会）；国家现代农业产业技术体系建设专项（CARS-45-19）；国家自然科学基金（31672690） |
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| 摘 要： | 针对CyHV-2病毒ORF121基因(GenBank:AFJ20543.1)进行原核表达系统的构建,将纯化重组蛋白作为抗原来免疫BALB/c小鼠获得多克隆抗体,应用该抗体开展CyHV-2病毒诊断及其感染机制研究。以CyHV-2病毒感染细胞上清液为扩增模板,扩增ORF121基因构建至pGEX-4T原核表达载体,经异丙基硫代半乳糖苷(IPTG)诱导表达rORF121重组蛋白,利用尿素纯化后免疫6周龄BALB/c小鼠制备多克隆抗体。结果显示,CyHV-2病毒ORF121基因可在原核表达系统中高效表达目的重组蛋白rORF121,经SDS-PAGE分析大小约为60 ku,主要以不可溶的包涵体存在。利用尿素溶解rORF121蛋白免疫BALB/c小鼠获得抗ORF121蛋白的多克隆抗体,Western Blot实验显示,该抗体可特异性识别CyHV-2病毒感染RyuF-2细胞样品。研究表明,利用CyHV-2感染RyuF-2细胞后,本研究制备的抗ORF121蛋白的多克隆抗体能够通过间接免疫荧光实验特异性识别CyHV-2病毒感染的细胞样品。本研究制备的抗ORF121蛋白的多克隆抗体,能够为CyHV-2病毒诊断技术的构建以及深入开展CyHV-2病毒感染机制提供良好的技术基础。
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| 关 键 词： | Ⅱ型鲤疱疹病毒 ORF121 原核表达 多克隆抗体 间接免疫荧光检测 |
| 收稿时间： | 2018-09-18 |
| 修稿时间： | 2019-01-23 |
Preparation and characterization of polyclonal antibody againstCyprinid herpesvirus 2 ORF121 |
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| YU Lin,L,#; Liqun and WANG Hao. Preparation and characterization of polyclonal antibody againstCyprinid herpesvirus 2 ORF121[J]. Journal of Fisheries of China, 2019, 43(6): 1463-1471 |
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| Authors: | YU Lin,L&# Liqun WANG Hao |
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| Affiliation: | National Pathogen Collection Center for Aquatic Animals, Shanghai Ocean University, Shanghai 201306, China,National Pathogen Collection Center for Aquatic Animals, Shanghai Ocean University, Shanghai 201306, China;Key Laboratory of Freshwater Aquatic Genetic Resources Aquaculture, Ministry of Agriculture and Rural Affairs, Shanghai Ocean University, Shanghai 201306, China;National Experimental Teaching Demonstration Center for Fishery Sciences, Shanghai Ocean University, Shanghai 201306, China and National Pathogen Collection Center for Aquatic Animals, Shanghai Ocean University, Shanghai 201306, China;Key Laboratory of Freshwater Aquatic Genetic Resources Aquaculture, Ministry of Agriculture and Rural Affairs, Shanghai Ocean University, Shanghai 201306, China;National Experimental Teaching Demonstration Center for Fishery Sciences, Shanghai Ocean University, Shanghai 201306, China |
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| Abstract: | Cyprinid herpesvirus 2 (CyHV-2) is an emerging pathogen of the crucian carp, Carassius carassius, which has caused huge economic losses in China and appears to be spreading worldwide. The objective of this study was to produce and characterize new polyclonal antibodies against CyHV-2 ORF121 that were suitable for diagnostic use and the investigation of ORF121 function. The open reading frame (ORF) of 121 was amplified from the culture supernatant of the CyHV-2. ORF121 was subcloned into the pGEX-4T vector and recombinant protein was expressed in Escherichia coli. The purified recombinant protein ORF121 was used to immunize mice and polyclonal antibodies were obtained. The recombinant protein ORF121 was purified under urea conditions then used to 6-week-old BALA/c mice to prepare its polyclonal antibody. Western Blot and indirect fluorescence assay (IFA) assay were used to validate the ORF121 polyclonal antibody. We found the recombinant protein ORF121(rORF121) could be expressed in E. coli. aggregated in the form of inclusion body. The specificity of the anti-ORF121 polyclonal antibody was confirmed by Western Blot. IFA with anti-ORF121 polyclonal antibody for the detection of CyHV-2 infected RyuF-2 cells was developed in this study. In conclusion, the anti-ORF121 polyclonal antibody will be a valuable tool in further studies to elucidate the mechanisms of viral infection and CyHV-2 diagnosis. |
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| Keywords: | Cyprinid herpesvirus 2 ORF121 prokaryotic expression polyclonal antibody IFA |
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