H9N2禽流感病毒HA蛋白mRNA疫苗的构建表达及鉴定  

Construction and expression and identification of H9N2 avian influenza virus HA protein mRNA vaccine

作　　者：花悦 宋奇珊 王东东 杨思敏 王颖[1] 任建乐 牛胜 尚桂军 赵宇军[1] 田文霞[1] HUA Yue;SONG Qishan;WANG Dongdong;YANG Simin;WANG Ying;REN Jianle;NIU Sheng;SHANG Guijun;ZHAO Yujun;TIAN Wenxia(College of Veterinary Medicine,Shanxi Agricultural University,Jinzhong 030801,China;Shanxi Key Laboratory of Protein Structure Determination,Shanxi Academy of Advanced Research and Innovation,Taiyuan 030032,China)

机构地区：[1]山西农业大学动物医学学院,山西晋中030801 [2]山西高等创新研究院功能蛋白结构解析山西省重点实验室,山西太原030032

出　　处：《中国兽医科学》2024年第5期609-614,共6页Chinese Veterinary Science

基　　金：中美疾病预防控制中心合作项目(5U01IP001106-04-00,2022HX116,E2900901-02);山西省重点研发项目(202102130501001);山西省现代农业产业技术体系建设专项资金(2021-07,2022-07,2023CYJSTX15-09);山西省科技创新青年人才团队项目(202204051001022);功能蛋白结构解析山西省重点实验室资助项目(202104010910006);山西省1331工程项目(20211331-13)。

摘　　要：H9N2亚型禽流感病毒(AIV)是严重危害家禽养殖业的病原,由于目前的疫苗无法应对突发的AIV疫情,因此本研究设计一种基于血凝素(HA)基因的抗H9N2亚型禽流感病毒mRNA疫苗。在原有Puc57克隆载体基础上,构建mRNA体外转录模板,将PCR得到的HA基因和EGFP基因插入模板中构建重组质粒Puc57-HA和Puc57-EGFP。将重组质粒线性化后,进行体外转录、加帽、加尾、纯化等一系列修饰并转染鸡成纤维细胞(DF-1),观察48 h荧光表达情况,并用Western-blot检测HA蛋白在细胞中的表达。结果显示,48 h出现绿色荧光,Western-blot显示HA蛋白成功表达。本试验为H9N2禽流感mRNA疫苗的研制奠定了基础。H9N2 avian influenza virus is one of the etiological agents that seriously threaten the poultry industry.As the current vaccine can not cope with the sudden AIV epidemic,this study designed an mRNA vaccine against H9N2 subtype avian influenza virus based on hemagglutinin(HA)gene.On the basis of the original Puc57 cloning vector,mRNA transcription template was constructed in vitro,and the HA gene and EGFP gene obtained by PCR were inserted into the template to construct the recombinant plasmid Puc57-HA and Puc57-EGFP.The recombinant plasmid was linearized,transcribed in vitro,capped,caudated and purified,and then transfected into chicken fibroblasts(DF-1)cells,fluorescence expression was observed for 48 h,and the expression of HA protein in cells was detected by Western-blot.The results showed that green fluorescence appeared at 48 h,and Western-blot showed that HA protein was successfully expressed.This experiment laid a foundation for the development of mRNA vaccine for H9N2 avian influenza.

关 键 词：禽流感 mRNA疫苗 体外转录 

分 类 号：S852.659.5[农业科学—基础兽医学]