N端B细胞表位缺失的兔出血症病毒VP60蛋白的表达及其免疫原性  

Expression of rabbit hemorrhagic disease virus VP60 protein with B cell epitope deletion in N terminus and its immunogenicity in rabbit

作　　者：刘维龙 胡波[1,3] 范志宇 魏后军[1,3] 仇汝龙 宋艳华[1,3] 陈萌萌 葛雷 熊富强[3,4] 王芳 LIU Wei-long;HU Bo;FAN Zhi-yu;WEI Hou-jun;QIU Ru-long;SONG Yan-hua;CHEN Meng-meng;GE Lei;XIONG Fu-qiang;WANG Fang(Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences/Key Laboratory for Veterinary Bio-Product Engineering,Ministry of Agriculture,Nanjing 210014,China;Xizang Agricultural and Animal Husbandry University,Linzhi 860000,China;GuoTai(Taizhou)Center of Technology Innovation for Veterinary Biologicals,Taizhou 225300,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]江苏省农业科学院兽医研究所/农业部兽用生物制品工程重点实验室,江苏南京210014 [2]西藏农牧学院,西藏林芝860000 [3]兽用生物制品<泰州>国泰技术创新中心,江苏泰州225300 [4]南京农业大学动物医学院,江苏南京210095

出　　处：《江苏农业学报》2024年第3期508-513,共6页Jiangsu Journal of Agricultural Sciences

基　　金：现代农业产业技术体系建设专项资金资助项目(CARS-43-C-1)。

摘　　要：本研究将A3C单抗识别的VP60蛋白NTA区域作为识别标记,设计一系列编码VP60 NTA区域重叠多肽的基因序列并连接于pGEX-4T-1载体,确定A3C单抗识别的精确表位。然后构建重组质粒Bacmid-VP60△A3C,将Bacmid-VP60△A3C转染Sf9昆虫细胞,得到重组杆状病毒rBac-VP60△A3C。RT-PCR、HA、IFA和Western Blot鉴定结果表明,重组蛋白VP60△A3C在Sf9细胞中高效表达,电镜观察显示其形态和结构与兔出血症病毒VP60蛋白类似。将重组蛋白VP60△A3C以每只200μg免疫2月龄RHDV血清阴性的新西兰兔,免疫后14 d,以兔出血症病毒WF株攻毒新西兰兔。结果表明,免疫组无死亡,而对照组全部死亡。本研究结果为制备表位缺失的兔出血症亚单位疫苗奠定了基础。This study used the NTA region of the VP60 protein recognized by A3C monoclonal antibody as a recognition marker,designed a series of gene sequences encoding overlapping peptides in the VP60 NTA region,and connected them to the pGEX-4T-1 vector to determine the precise epitopes recognized by A3C monoclonal antibody.Then recombinant plasmid Bacmid-VP60△A3C was constructed.Bacmid-VP60△A3C was transfected into Sf9 insect cells,and recombinant baculovirus rBac-VP60△A3C was obtained.The identification results of RT-PCR,HA,IFA,and Western blot showed that the recombinant protein VP60△A3C was highly expressed in Sf9 cells.Electron microscopy observation showed that its morphology and structure were similar to those of rabbit hemorrhagic disease virus VP60 protein.The 2-month-old RHDV serum negative New Zealand rabbits were immunized with recombinant protein VP60△A3C at a rate of 200μg per rabbit,and New Zealand rabbits were challenged with rabbit hemorrhagic disease virus WF strain after 14 days.The results showed that there were no deaths in the immune group,while all deaths occurred in the control group.The results of this study lay the foundation for the development of epitope-deleted subunit vaccine of rabbit hemorrhagic disease.

关 键 词：兔出血症病毒 VP60蛋白 B细胞表位 免疫原性 

分 类 号：S855.3[农业科学—临床兽医学]