单壁碳纳米管载锦鲤疱疹病毒ORF149核酸疫苗的构建  

Construction of Single-walled Carbon Nanotube-loaded Koi Herpes Virus ORF149 Nucleic Acid Vaccine

作　　者：胡峰 王庆[1] 李莹莹[1] 曾伟伟[1] 王高学[3] 朱斌[3] 王英英[1] 尹纪元 HU Feng;WANG Qing;LI Ying-ying;ZENG Wei-wei;WANG Gao-xue;ZHU Bin;WANG Ying-ying;YIN Ji-yuan(Key Laboratory of Fishery Drug Development of Ministry of Agriculture,Key Laboratory of Aquatic Animal Immune Technology of Guangdong Province,Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510380;College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306;College of Animal Science and Technology,Northwest A&F University,Yangling 712100)

机构地区：[1]中国水产科学研究院珠江水产研究所农业部渔药创制重点实验室广东水产动物免疫技术重点实验室,广州510380 [2]上海海洋大学水产与生命学院,上海201306 [3]西北农林科技大学动物科技学院,杨凌712100

出　　处：《生物技术通报》2020年第2期206-213,共8页Biotechnology Bulletin

基　　金：广东省自然科学基金项目（2018A0303130029）;中国水产科学研究院珠江水产研究所基本科研业务费（2019ZX-002）;现代农业产业技术体系建设专项资金（CARS-45）

摘　　要：为了开发新型高效疫苗预防锦鲤疱疹病毒病,以单壁碳纳米管(SWCNT)作为运输载体,构建锦鲤疱疹病毒(KHV)ORF149核酸疫苗。首先构建pcDNA3.1(+)-ORF149重组质粒,通过瞬时转染和免疫印迹分析确定其表达情况,然后通过1,3-偶极环加成反应法将重组质粒与SWCNTs进行偶联,最后通过扫描电镜观察和核酸电泳鉴定其偶联是否成功。结果表明,构建的重组质粒转染细胞后经免疫印迹分析和间接免疫荧光试验均能检测到特异性信号;在核酸琼脂糖凝胶电泳中,构建的重组疫苗电泳条带消失;在场发射扫描电镜观察下,与重组质粒进行偶联的SWCNTs和空白SWCNTs形态差异明显。In order to develop a new type of highly effective vaccine against Koi Herpesvirus(KHV)disease,a single-walled carbon nanotube(SWCNT)was used as a transport vector to construct a KHV ORF149 nucleic acid vaccine. The recombinant plasmid pcDNA3.1(+)-ORF149 was firstly constructed,and its expression was confirmed by transient transfection and immunoblot analysis. Then,the recombinant plasmid was conjugated to SWCNTs by 1,3-dipolar cycloaddition reaction. Scanning electron microscopy and nucleic acid electrophoresis was conducted to identify whether the conjugation was successful. The results showed that specific signals were detected with constructed recombinant plasmid in both immunoblot analysis and indirect immunofluorescence assay after transfection. In the nucleic acid agarose gel electrophoresis,the electrophoretic band of the constructed recombinant vaccine disappeared. Under the observation of scanning electron microscopy,the morphologies of the conjugated SWCNTs with the recombinant plasmid and blank SWCNTs were significantly different.

关 键 词：单壁碳纳米管 KHV ORF149 DNA疫苗 

分 类 号：S943[农业科学—水产养殖]