A型口蹄疫病毒AF72株病毒样颗粒制备条件优化及鉴定  

Optimization of preparation conditions and identification of virus-like particles of AF72 strain of foot-and-mouth disease virus type A

作　　者：张雅雅 李昊洲 王运航 王田田 李河林 黄增文 孙世琪[2] 郭慧琛[2] 毕台飞[1] 敬晓棋[1] ZHANG Ya-ya;LI Hao-zhou;WANG Yun-hang;WANG Tian-tian;LI He-lin;HUANG Zeng-wen;SUN Shi-qi;GUO Hui-chen;BI Tai-fei;JING Xiao-qi(School of Life Sciences,Yulin University,Yulin 719000,China;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;Xichang University,Xichang 615000,China)

机构地区：[1]榆林学院生命科学学院,陕西榆林719000 [2]中国农业科学院兰州兽医研究所,甘肃兰州730046 [3]西昌学院,四川西昌615000

出　　处：《中国兽医科学》2023年第12期1507-1515,共9页Chinese Veterinary Science

基　　金：榆林市产学研科技项目(CXY-2021-87);陕西省科技厅一般项目(S2023-YF-YBNY-0080);陕西省重点研发计划项目(2023-YBNY-143)。

摘　　要：为了制备A型口蹄疫病毒AF72株病毒样颗粒,本研究通过优化IPTG浓度、诱导时菌液D600值、诱导温度、诱导时间来提高目标蛋白可溶性表达量,在特定酶切组装液中组装颗粒并调整组装液成分以提高其在体外的组装率,利用SDS-PAGE、蔗糖密度梯度离心、动态光散射、DAS-ELISA、透射电镜等多种方法对其进行鉴定。结果显示,经SDS-PAGE鉴定,VP0、VP1、VP3三条带均存在且符合目的条带大小,纳米粒径在30 nm左右符合口蹄疫病毒粒子大小,DAS-ELISA鉴定VP0、VP1、VP3在体外成功组装成病毒样颗粒,电子显微镜也拍出部分病毒样颗粒。上述结果表明,A型口蹄疫AF72株病毒样颗粒成功制备,为后续建立检测口蹄疫病毒中和抗体的液相阻断ELISA奠定了基础。To prepare virus-like particles of AF72 strain of FMDV type A,this study improved the soluble expression of the target protein by optimizing IPTG concentration,Doo value of bacterial solu-tion at induction,induction temperature,and induction time.The particles were assembled in a specific enzyme-cut assembly solution and the composition of the assembly solution was adjusted to improve its assembly rate in vitro.Subsequently,SDS-PAGE,sucrose density gradient centrifugation,dynamic light scattering,DAS-ELISA,and transmission electron microscopy were used to determine target protein.The results showed that the three bands of VPO,VP1 and VP3 were identified by SDS-PAGE and were consistent with the size of the target band.The particle size of the nanoparticle was about 30 nm and was consis-tent with the size of the virus particles of foot-and-mouth disease.VPo,VPl and VP3 was identified by DAS-ELISA and successfully assembled into virus-like particles in vitro,and some virus-like particles were also photographed by electron microscope.The above results indicated that virus-like particle of AF72 strain of FMDV type A were successfully prepared,which laid a foundation for establishing a liquidphase blocking ELISA for detecting FMDV neutralizing antibody.

关 键 词：A型口蹄疫 AF72株 VP0、VP1、VP3融合表达 病毒样颗粒组装鉴定 

分 类 号：S852.659.6[农业科学—基础兽医学]