D型流感病毒NP蛋白的原核表达及多克隆抗体的制备  

Prokaryotic expression of NP protein of influenza D virus and preparation of polyclonal antibody

作　　者：肖雨晴 程娇娇 孙锐瑶 陈妍希 谯薇美 尹鑫 远立国 李守军 卢刚 XIAO Yuqing;CHENG Jiaojiao;SUN Ruiyao;CHEN Yanxi;QIAO Weimei;YIN Xin;YUANLiguo;LI Shoujun;LU Gang(Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases,College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China;Harbin Veterinary Research Institute,Chinese AcademyofAgricultural Sciences,Harbin150069,China)

机构地区：[1]华南农业大学兽医学院,广东省兽医临床重大疾病综合防控重点实验室,广东广州510642 [2]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150069

出　　处：《中国兽医学报》2023年第10期2035-2041,共7页Chinese Journal of Veterinary Science

基　　金：黑龙江省博士后科研启动金资助项目(LBH-Q21199)。

摘　　要：该研究旨在对D型流感病毒(influenza D virus,IDV)NP蛋白进行原核表达并制备多克隆抗体,为D型流感快速诊断技术奠定基础。采用PCR技术扩增D/bovine/Mississippi/c00046N/2014毒株的NP片段编码区,并对其序列进行跨膜区、信号肽、疏水键、抗原性及结构域分析,筛选出最优区段以制备多克隆抗体。该研究将IDV NP最优编码区序列插入原核表达载体pET-B2M,构建pET-B2M-NP重组阳性质粒,经PCR和测序鉴定后将重组阳性质粒转化至大肠杆菌TreliefTM5α中;IPTG诱导表达后采用镍柱亲和层析法纯化重组NP蛋白,并免疫新西兰白兔制备多克隆抗体。序列分析结果显示,1~500氨基酸区段无跨膜区和信号肽序列,局部亲水性好且具备良好的免疫原性,为制备重组NP蛋白的最佳抗原区段。经测序验证构建了pET-B2M-NP重组阳性质粒,转化后经IPTG诱导,主要以包涵体的形式表达了相对分子质量大小约为72 kDa的蛋白,符合预期。间接ELISA结果显示,制备的多克隆抗体效价达1∶100000且免疫原性良好。Western blot和间接免疫荧光试验结果均表明,所制备的NP多克隆抗体能高效地与IDV发生特异性结合。该研究通过原核表达系统表达了IDV重组NP蛋白,制备了免疫原性和特异性良好的兔抗NP多克隆抗体,为研究NP蛋白的结构和功能提供了条件,为揭示IDV致病机制奠定了基础。The purpose of this study was to express NP protein of influenza D virus(IDV)using prokaryotic expression system and prepare polyclonal antibodies to lay the foundation for rapid diagnosis of influenza D.PCR was used to amplify the NP fragment coding region of the D/bovine/Mississippi/c00046N/2014 strain,and the transmembrane region,signal peptide,hydrophobic bond,antigenicity and structural domain of the sequence were analyzed,so as to screen the optimal region for the preparation of polyclonal antibody.The recombinant NP protein was purified by nickel column affinity chromatography after IPTG induction,and polyclonal antibodies were prepared by immunizing New Zealand white rabbits.The sequence analysis results showed that the 1-500 amino acid region had no transmembrane region and signal peptide sequence,and had good local hydrophilicity and immunogenicity,which was the best antigen region for preparing recombinant NP protein.The recombinant positive plasmid pET-B2M-NP was verified by sequencing.After transformation,it was induced by IPTG and expressed a protein with a molecular mass of about 72 kDa mainly in the form of inclusion bodies,which was in line with the expectation.The results of indirect ELISA showed that the titer of the polyclonal antibody was 1:100000 and had a good immunogenicity.The results of Western blot and indirect immunofluorescence showed that the NP polyclonal antibody could bind specifically to IDV with high efficiency.The recombinant IDV NP protein was expressed in prokaryotic expression system,and the rabbit anti-NP polyclonal antibodies with good immunogenicity and specificity were prepared,which provided conditions for studying the structure and function of NP protein and laid a foundation for investigating the pathogenic mechanism of IDV.

关 键 词：D型流感病毒 NP蛋白 原核表达 多克隆抗体 

分 类 号：S855.3[农业科学—临床兽医学]