兔出血症病毒VP60蛋白在杆状病毒表达系统中的表达优化  

OPTIMIZATION OF RABBIT HEMORRHAGIC VIRUS VP60 PROTEIN PRODUCTION IN THE BACULOVIRUS EXPRESSION SYSTEM

作　　者：张帅 谭菲菲 王妍 宋欢欢 李向东 王同燕 田克恭 ZHANG Shuai;TAN Fei-fei;WANG Yan;SONG Huan-huan;LI Xiang-dong;WANG Tong-yan;TIAN Ke-gong(College of Animal Science and Veterinary Medicine,Henan Agricultural University,Zhengzhou 450000,China;National Research Center for Wterinary Medicine,Luoyang 471003,China)

机构地区：[1]河南农业大学牧医工程学院,郑州450000 [2]国家兽用药品工程技术研究中心,洛阳471003

出　　处：《中国动物传染病学报》2020年第6期33-41,共9页Chinese Journal of Animal Infectious Diseases

基　　金：郑洛新国家自主创新示范区首批创新引领型产业集群专项:畜禽用安全高效新型疫苗研发及产业化(181200211700)。

摘　　要：为提高兔出血症病毒衣壳蛋白VP60在Bac-to-Bac系统的表达量,本研究使用Hr1、Hr3、WPRE、SV40、CAG、Melt等顺式作用元件及元件组合对pFastBacDual供体质粒进行改造,将改造后的供体质粒用于重组VP60蛋白的表达。通过重组杆状病毒滴度测定、IFA、SDS-PAGE、Western blot、血凝效价测定等方法对重组VP60蛋白的产量、反应原性进行评价。结果表明,所有重组VP60蛋白均能够与VP60单克隆抗体反应,具有良好的反应原性,Hr1、Hr3、SV40、Hr1-WPRE、Hr3-WPRE、SV40-WPRE、SV40-WPRE-Melt能够提高重组VP60蛋白产量,其中Hr3、SV40-WPRE改造类型的提升作用更为显著,研究结果为降低兔出血症病毒亚单位疫苗生产成本提供参考,对其他重组蛋白在Bac-to-Bac系统中的表达量提升具有一定的借鉴意义。In order to increase the expression level of the Rabbit hemorrhagic disease virus capsid protein VP60 in the Bac-to-Bac system,the molecular cloning technology was utilized in this study to modify the pFastBacDual donor plasmid with cis-acting elements and elements combinations of Hr1,Hr3,WPRE,SV40,CAG,Melt,etc.The modified donor plasmids were used for the production of recombinant VP60 protein.The yields of the recombinant VP60 proteins were evaluated by titration of recombinant Baculoviruses,IFA,SDS-PAGE,Western blot and hemagglutination.The results showed that the recombinant VP60 protein reacted with VP60 MAb and Hr1,Hr3,SV40,Hr1-WPRE,Hr3-WPRE,SV40-WPRE,SV40-WPRE-Melt increased the yield of recombinant VP60 protein,among which Hr3 and SV40-WPRE showed the best effect.Our research results provided a reference for the production of Rabbit hemorrhagic disease virus subunit vaccine at a lower cost,which may be used for the expression of other recombinant proteins.

关 键 词：兔出血症病毒 VP60蛋白 杆状病毒 表达优化 

分 类 号：S852.659.1[农业科学—基础兽医学]