蓝舌病病毒VP7蛋白的可溶性纯化及多克隆抗体的制备  

Soluble expression,purification and polyclonal antibody preparation of bluetongue virus VP7 protein

作　　者：丁晓攀 车永军 郭艳妮 高闪电[1] 田占成[1] ALI MOHAMED Darien Kheder 关贵全[1] 孙世琪[1] 殷宏[1,2] 独军政 DING Xiao-pan;CHE Yong-jun;GUO Yan-ni;GAO Shan-dian;TIAN Zhan-cheng;ALI MOHAMED Darien Kheder;GUAN Gui-quan;SUN Shi-qi;YIN Hong;DU Jun-zheng(State Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,甘肃兰州730046 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2021年第9期1128-1133,共6页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31672562);国家重点研发计划项目(2017YFD0502304)。

摘　　要：首先参照GenBank中已发表的基因序列人工合成蓝舌病病毒VP7基因,将其克隆到表达载体pSMK上。选取含有正确开放阅读框的重组质粒pSMK-VP7,转化大肠杆菌BL21感受态细胞,于16℃利用IPTG诱导剂进行诱导表达,优化表达条件,纯化重组VP7蛋白,免疫新西兰白兔制备VP7多克隆抗体,利用免疫印迹试验对制备的多克隆抗体进行特异性分析。结果显示,重组VP7蛋白在大肠杆菌中呈可溶性表达,且16 h表达量最大,利用镍离子树脂纯化获得了高纯度的VP7蛋白;原核表达的重组VP7以及天然的VP7蛋白均能够与VP7多克隆抗体发生特异性反应。本研究成功实现了重组BTV VP7蛋白的可溶性表达和纯化,并制备了特异性良好的多克隆抗体,为进一步开展BTV的VP7功能研究和建立BTV诊断方法奠定了基础。The VP7 gene of bluetongue virus(BTV)was artificially synthesized by referring to the published BTV gene sequence in GenBank,and cloned into the expression vector pSMK.The expression plasmid p SMK-VP7 containing the correct open reading frame was transformed into competent cells E.coli BL21 and induced with IPTG at 16℃.The expression parameters were optimized and the recombinant VP7 proteins were purified.New Zealand white rabbits were immunized to prepare the VP7 polyclonal antibodies.Immunoblot assay was used to analyze the specificity of prepared polyclonal antibodies.The results showed that the recombinant VP7 protein was soluble in E.coli and had the largest amount of induction in 16 h.High-purity VP7 protein was obtained by purification using nickel ion resin.The recombinant expressed VP7 and natural VP7 proteins can both react specifically with VP7 polyclonal antibodies.In this study,the recombinant VP7 protein of BTV was expressed in soluble form and purified,and the polyclonal antibodies against VP7 protein was prepared,which will lay the foundation for further research on function of VP7 protein and diagnostic methods of BTV.

关 键 词：蓝舌病病毒 VP7基因 可溶性表达 纯化 多克隆抗体 

分 类 号：S852.6594[农业科学—基础兽医学]