马疱疹病毒8型gD蛋白的生物信息学分析及多克隆抗体制备  

Bioinformatic Analysis and Polyclonal Antibody Preparation of Equine Herpesvirus Type 8 gD Protein

作　　者：徐萌 郗灿坤 王天娇 李瑞博 丁相丹 孙祺 胡乐玉 任慧英[2] 李亮亮 王彤彤 XU Meng;XI Can-kun;WANG Tian-jiao;LI Rui-bo;DING Xiang-dan;SUN Qi;HU Le-yu;REN Hui-ying;LI Liang-liang;WANG Tong-tong(College of Agronomy and Agricultural Engineering,Liaocheng University,Liaocheng,Shandong,252000,China;College of Veterinary Medicine,Qingdao Agricultural University,Qingdao,Shandong,266109,China)

机构地区：[1]聊城大学毛驴高效繁育与生态饲养研究院,山东聊城252000 [2]青岛农业大学动物医学院,山东青岛266109

出　　处：《动物医学进展》2023年第8期11-16,共6页Progress In Veterinary Medicine

基　　金：聊城大学大学生创新项目(CXCY2022121,CXCY2022250,CYCX2022434);国家自然科学基金(32002248);山东省自然科学基金(ZR2020QC016,ZR2020QC017)。

摘　　要：旨在探索马疱疹病毒8型(Equine herpesvirus type 8,EHV-8)囊膜蛋白gD的生物信息学特性、原核表达和多克隆抗体制备。用生物信息学分析软件对gD基因编码蛋白质的理化性质、亲/疏水性和跨膜区进行分析,以EHV-8 SDLC66毒株为模板,通过PCR扩增gD蛋白的胞外区并克隆至pET-32a(+)载体中,将重组质粒pET-32a-gD转化至BL21(DE3)中,经IPTG诱导表达后,收集菌体超声破碎,纯化并获得重组gD蛋白,免疫新西兰兔制备多克隆抗体,用间接ELISA测定所制备多克隆抗体的效价,同时用Western blot与间接免疫荧光试验(IFA)明确所制备多克隆抗体的特异性。结果表明,EHV-8 gD编码的蛋白稳定,是亲水性蛋白,有跨膜区,位于30-330 aa。gD蛋白跨膜区正确克隆入pET-32a(+)载体,获得重组质粒pET-32a-gD。重组gD蛋白在大肠埃希氏菌BL21(DE3)中以包涵体形式表达,分子质量约为55 ku,将其纯化后免疫新西兰兔获得抗gD多克隆抗体,该抗体的效价高达1∶200 000,Western blot与IFA结果表明该多克隆抗体具有良好的特异性。The aim of present study was to analyze bioinformation,expressing and preparation of polyclonal antibodies against EHV-8 gD protein.Firstly,physicochemical properties,hydrophilia and hydrophobicity,and transmembrane domain were analyzed with bioinformatics analysis software.The extracellular region of gD was amplified with SDLC66 strain genome,and cloned into pET-32a(+)vector.Further,the recombinant plasmid was transformed into BL21(DE3)and induced with IPTG for expression.The recombinant gD protein after ultrasonication was purified and used to immunize rabbits to prepare polyclonal antibodies.The titer of polyclonal antibodies was determined by ELISA,the specificity of polyclonal antibodies was analyzed by Western blot and indirect immunofluorescence assay(IFA).These results showed that the gD protein was stable,hydrophilic protein and has transmembrane domain(30-330 aa).The transmembrane domain of gD protein was cloned into pET-32a(+)vector,and obtained the recombinant plasmid pET-32a-gD.The recombinant gD protein was inclusion bodies and its molecular weight is about 55 ku.The titer of the polyclonal antibodies was 1∶200000.The results of Western blot and IFA suggested that the polyclonal antibodies of EHV-8 gD protein was specific.

关 键 词：马疱疹病毒8型 囊膜蛋白gD 生物信息学分析 多克隆抗体 原核表达 

分 类 号：S852.652[农业科学—基础兽医学]