基于GP64单抗的重组杆状病毒滴度测定方法的建立及应用  

Establishment and application of titer determination method based on GP64 monoclonal antibody for recombinant baculovirus

作　　者：王妍 邓跃 宋欢欢 贺笋 张超林 李俊辉 颜世君 王尊宝 王同燕 谭菲菲 田克恭 WANG Yan;DENG Yue;SONG Huan-huan;HE Sun;ZHANG Chao-lin;LI Jun-hui;YAN Shijun;WANG Zun-bao;WANG Tong-yan;TAN Fei-fei;TIAN Ke-gong(College of Animal Science and Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;Nutional Research Center for Veterinary Medicitie,Luoyang,Henan 471000,China;Pulike Biological Engineering Inc,Luoyang,Henan 471000,China;Tecon Bio-technology Co.,Ltd.,Urumqi 830011,China)

机构地区：[1]河南农业大学牧医工程学院,河南郑州450046 [2]国家兽用药品工程技术研究中心,河南洛阳471000 [3]普莱柯生物工程股份有限公司,河南洛阳471000 [4]天康生物股份有限公司,新疆乌鲁木齐830011

出　　处：《中国兽医学报》2020年第11期2090-2096,共7页Chinese Journal of Veterinary Science

基　　金：郑洛新国家自主创新示范区创新引领型产业集群资助项目(181200211700)。

摘　　要：杆状病毒表达系统是表达重组蛋白的首选真核表达系统之一,该系统通过表达目标蛋白的重组杆状病毒感染昆虫细胞,可以产生大量的生物活性蛋白。在重组杆状病毒生产过程中,为了获得高表达的重组蛋白,需要准确地测定病毒滴度。当前已有的滴度测定方法耗时较长,或仅限于表达报告基因的病毒。本研究利用前期获得的GP64单克隆抗体,针对表达猪细小病毒(porcine parvovirus virus,PPV)VP2蛋白的重组杆状病毒进行滴度测定,通过对GP64单抗、HRP羊抗鼠二抗、山羊血清稀释比例的优化,初步建立了一种杆状病毒滴度测定方法。结果显示,GP64单抗的最佳稀释比例为1∶500~1∶1000,二抗的最佳稀释比例为1∶250~1∶500,山羊血清的最佳稀释比例为1∶30~1∶50。该方法测定的5种不同病毒滴度与商品化试剂盒测定的滴度差异不显著,表明初步建立了一种高效稳定的杆状病毒滴度测定方法,为杆状病毒表达系统生产重组蛋白奠定基础。Baculovirus expression system is the preferred system for recombinant protein expression.Large amounts of bioactive protein can be generated using this system by infecting insect cells.In order to obtain highly expressed recombinant protein during the production of recombinant baculovirus,it is necessary to accurately determine the virus titer.Existing titer determination methods are time-consuming or are limited to virus expressing reporter genes.In this study,the GP64 monoclonal antibody obtained early was used to determine the titer of recombinant baculovirus expressingVP2 protein of porcine parvovirus virus(PPV).The dilution ratio of GP64 monoclonal antibody,HRP goat anti-mouse antibody and goat serum was optimized,and a baculovirus titer determination method was initially established.The results showed that the optimal dilution range of GP64 monoclonal antibody is 1∶500-1∶1000,the optimal dilution range of secondary antibody is 1∶250-1∶500,and the optimal dilution range of goat serum is 1∶30-1∶50.The five different virus titers determined by this method are not significantly different from the titers determined by commercial kits.It shows that a highly efficient and stable baculovirus titer determination method has been successfully established,which lays the foundation for the production of recombinant proteins by the baculovirus expression system.

关 键 词：杆状病毒 GP64单抗 猪细小病毒 VP2蛋白 病毒滴度 

分 类 号：S852.65[农业科学—基础兽医学]