水疱性口炎病毒核蛋白的纳米抗体和多聚抗核蛋白纳米抗体的原核表达及功能验证  

Prokaryotic expression and functional verification of nanoantibodies and poly-nanoantibodies against nucleoprotein of vesicular stomatitis virus

作　　者：郭玉堃 马英先 常雯茹 明胜利 杨国宇[1] 郭豫杰[1] GUO Yukun;MA Yingxian;CHANG Wenru;MING Shengli;YANG Guoyu;GUO Yujie(Key Laboratory of Animal Biochemistry and Nutrition,Ministry of Agriculture and Rural Affairs,Henan Agricultural University,Zhengzhou 450046,China)

机构地区：[1]河南农业大学农业农村部动物生物化学与营养重点开放实验室,河南郑州450046

出　　处：《中国兽医学报》2021年第9期1762-1769,共8页Chinese Journal of Veterinary Science

基　　金：动物重大疫病新概念防控产品研发资助项目(2017YFD0501100);河南省高等学校重点科研资助项目(17A230013)。

摘　　要：分别制备出高可溶性的抗水疱性口炎病毒(vesicular stomatitis virus,VSV)核蛋白纳米抗体VSVNb和重组截短型铁蛋白与抗VSV核蛋白串联的多聚纳米抗体的融合蛋白FnL-VSV,并对融合蛋白VSVNb和FnL-VSV在VSV诊断中的应用进行了初步探索。从羊驼(Vicugna pacos)中获得抗VSV核蛋白的纳米抗体序列,命名为VSVNb;从激烈火球菌(Pyrococcus furiosus)中分离出来的铁蛋白(encapsulin)进行截短后,和从羊驼中获得的抗VSV核蛋白的纳米抗体序列进行串联,命名为FnL-VSV。载体构建后用大肠杆菌表达系统进行融合蛋白的原核表达,经Ni2+纯化后获得单一纳米抗体VSVNb;经硫酸铵盐析纯化得到单一融合蛋白FnL-VSV,并进行电镜检测。将制备的重组蛋白VSVNb和FnL-VSV分别进行HRP标记后,和VSV毒株用作直接ELISA试验。结果表明,原核表达与纯化后获得了纯度较高的融合蛋白VSVNb和FnL-VSV;电镜结果显示,重组蛋白FnL-VSV自组装形成了直径为20 nm纳米样颗粒;直接ELISA检测结果表明,在抗原浓度一定的条件下,重组蛋白VSVNb和FnL-VSV均与特异性抗原VSV毒株在较低浓度条件下结合,具有较高活性,且重组蛋白FnL-VSV比VSVNb D450 nm数值更大;而在重组蛋白VSVNb和FnL-VSV浓度一定的条件下,其能敏感的识别低浓度到高浓度区间的特异性抗原。本试验建立了稳定获得重组蛋白VSVNb和FnL-VSV重组蛋白质的方法,并初步探索了其在直接ELISA中的使用。成功制备了重组蛋白VSVNb和FnL-VSV,为进一步研究纳米抗体在VSV的诊断及后续抗病毒药物的研究奠定了基础。The purpose of this experiment was to prepare a highly soluble anti-vesicular stomatitis virus nuclear protein nano-antibody VSVNb and fusion protein FnL-VSV which was recombinant truncated ferritin in tandem with nano-antibodies against vesicular stomatitis virus nucleoprotein,and explore the application of fusion protein VSVNb and FnL-VSV in the diagnosis of VSV.Firstly,the anti-vesicular stomatitis virus N VHH sequence was obtained from Vicugna pacos and named VSVNb;the encapsulin isolated from Pyrococcus furiosus was truncated and concatenated with the VSVNb and named FnL-VSV.Then,the vector was constructed and the fusion protein was expressed by E.coli expression system.After Ni 2+purification,the nano antibody VSVNb was obtained and the fusion protein FnL-VSV was obtained by ammonium sulfate precipitation.VS-VNb and FnL-VSV were detected by electron microscope.Finally,the prepared recombinant protein VSVNb and FnL-VSV were labeled with HRP and evaluated by direct ELISA test.The results showed that VSVNb and FnL-VSV were obtained with high purity.The electron microscopy results showed that VSVNb and FnL-VSV formed nanoparticles with a diameter of 20nm.The ELISA results showed that at constant antigen concentration,VSVNb and FnL-VSV could recognize VSV with higher activity at a lower concentration,the D450nm value of recombinant protein FnL-VSV was higher than that of VSVNb.VSVNb and FnL-VSV could identify the specific antigen in the concentration range.In conclusion,we have established the method for VSVNb and FnL-VSV preparation and explored its use in VSV recognition by direct ELISA.The successful preparation of recombinant protein VSVNb and FnL-VSV laid an experimental foundation for the further study of the diagnosis of nano-antibodies in VSV and the subsequent study of antiviral drugs.

关 键 词：水疱性口炎病毒 核蛋白 纳米抗体 铁蛋白 直接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]