志贺氏杆菌分泌蛋白Dnak的表达纯化与多克隆抗体的制备及应用  

Expression and purification of Shigella secreted protein Dnak,preparation and application of polyclonal antibody

作　　者：贾雪娇 刘梦琦 刘洋 刘长城 李小凤 胡屹硕 李香雨 李润林 李永刚 赵微 JIA Xue-jiao;LIU Meng-qi;LIU Yang;LIU Chang-cheng;LI Xiao-feng;HU Yi-shuo;LI Xiang-yu;LI Run-lin;LI Yong-gang;ZHAO Wei(Laboratory of Pathogenic Biology,School of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121001,China)

机构地区：[1]锦州医科大学基础医学院病原生物学实验室,锦州121001

出　　处：《中国人兽共患病学报》2024年第3期236-242,共7页Chinese Journal of Zoonoses

基　　金：辽宁省科技厅自然科学基金面上项目(No.2021-MS-334);辽宁省教育厅面上项目基金(No.LJKMZ20221243)。

摘　　要：目的表达轮状病毒感染的乳鼠体内志贺氏杆菌分泌蛋白Dnak(Hsp70),并制备Dnak多克隆抗体。方法以提取的志贺氏杆菌基因组为模板PCR扩增Dnak片段,与pET-24a(+)和PCDNA3.1分别连接构建原核表达质粒pET24a(+)-Dnak和真核表达质粒PCDNA3.1-Dnak;pET24a(+)-Dnak原核表达质粒转化至E.coli BL21(DE3)感受态细胞,IPTG诱导表达,经镍柱纯化Dnak蛋白。以纯化后的重组蛋白为免疫原免疫C57BL/6小鼠制备多克隆抗体,采用间接ELISA检测效价,Western Blot法检测灵敏度和特异性。利用制备的多克隆抗体在GST Pull-down实验中检测Dnak与轮状病毒非结构蛋白Nsp2的相互作用。真核表达质粒PCDNA3.1-Dnak转染至HEK293T细胞,以制备的DnaK多克隆抗体为一抗,Western blot法检测Dnak在细胞中的表达。利用MEGA11及Genedoc软件分析此志贺氏杆菌与志贺菌属其他细菌Dnak氨基酸序列同源性。结果重组质粒pET24a(+)-Dnak与PCDNA3.1-Dnak经测序比对包含与此志贺氏杆菌(Shigella:PRJNA804371)序列一致的基因,证明质粒构建成功。诱导表达的重组Dnak蛋白主要以可溶性形式存在,相对分子质量约为75 kDa,浓度可达0.62 mg/mL;Dnak鼠多克隆抗体可与重组Dnak蛋白发生特异性反应,效价为1∶32000,至少可以在1∶6000稀释度下检测到Dnak蛋白。GST Pull-down实验可以检测到Dnak与轮状病毒非结构蛋白Nsp2发生相互作用。转染PCDNA3.1-Dnak重组质粒的HEK293T细胞可以表达Dnak蛋白。经MEGA11和Genedoc软件比对PRJNA804371株与宋内志贺菌(Shigella sonnei)、痢疾志贺菌(Shigella dysenteriae)、鲍氏志贺菌(Shigella boydii)、福氏志贺菌(Shigella flexneri)Dnak氨基酸序列同源性较高。结论成功表达志贺氏杆菌Dnak蛋白,具有良好反应性与免疫原性;制备的鼠多克隆抗体效价及灵敏度较高可满足后续实验需要。The purpose of this study was to express the secreted Shigella protein Dnak(Hsp70)in rotavirus-infected suckling mice,and to prepare Dnak polyclonal antibodies.The extracted Shigella genome was used as a template to amplify the Dnak fragment by PCR.The prokaryotic expression plasmid pET24a(+)-Dnak and eukaryotic expression plasmid PCDNA3.1-Dnak were ligated with pET-24a(+)and PCDNA3.1,respectively,and the pET24a(+)-Dnak prokaryotic expression plasmid was transformed into E.coli BL21(DE3)competent cells.After IPTG-induced expression,Dnak protein was purified with a nickel column.Polyclonal antibodies were prepared from the puri fied recombinant protein for immunogenic C57BL/6 mice,and the antibody titer was detected by indirect ELISA.Antibody sensitivity and specificity were detected by western blotting.The interaction between Dnak and rotavirus non-structural protein Nsp2 was detected by GST pull-down assay.The eukaryotic expression plasmid PCDNA3.1-Dnak was transfected into HEK293T cells,and the prepared Dnak polyclonal antibodies were used to detect the expression of Dnak in cells by western blotting.MEGA11 and Genedoc software were used to analyze the amino acid sequence homology between Shigella and other bacteria of the genus Shigella.The recombinant plasmids pET24a(+)-Dnak and PCDNA3.1-Dnak were sequenced and found to contain genes consistent with the sequence of Shigella:PRJNA804371,thus demonstrating successful plasmid construction.The induced expression of recombinant Dnak protein resulted primarily in soluble forms with a relative molecular weight of approximately 75 kDa;a concentration of 0.62 mg/ml;and good reactivity and immunogenicity.Dnak mouse polyclonal antibodies reacted specifically with recombinant Dnak protein at a titer of 1∶32000,and the limit of Dnak protein detection was a dilution of 1∶6000,thus indicating high titer and sensitivity to meet experimental needs.GST Pull-down assay can detect the interaction of Dnak with the rotavirus non-structural protein Nsp2.HEK293T cells transfecte

关 键 词：志贺氏杆菌 DNAK 蛋白表达与纯化 多克隆抗体 轮状病毒 

分 类 号：R373.9[医药卫生—病原生物学]