SARS-CoV-2 E蛋白表达及其多克隆抗体的制备  

Expression of a SARS-CoV-2 envelope protein and preparation of polyclonal antibodies against the SARS-CoV-2 envelope protein

作　　者：杜昆朋 陈柯 闫硕 高岩 徐海山 郑皖怿 李猛[1] 付玉荣[2] 伊正君[1] DU Kun-peng;CHEN Ke;YAN Shuo;GAO Yan;XU Hai-shan;ZHENG Wan-yi;LI Meng;FU Yu-rong;YI Zheng-jun(School of Medical Laboratory,Weifang Medical University,Wei fang,Shandong 261053,China;School of Basic Medicine,Weifang Medical Universiry)

机构地区：[1]潍坊医学院医学检验学院,山东潍坊261053 [2]潍坊医学院基础医学院

出　　处：《中国病原生物学杂志》2021年第12期1363-1366,1372,共5页Journal of Pathogen Biology

基　　金：山东省自然科学基金重点项目(No.ZR2018ZC1054)。

摘　　要：目的制备新型冠状病毒E蛋白和抗E蛋白多克隆抗体。方法原核质粒pMAT9s-2019-nCoV-E经鉴定验证正确后,转化BL21感受态细胞,IPTG诱导表达目的蛋白,经尿素梯度复性、Ni柱层析获得纯化的重组E蛋白,免疫兔获抗E多克隆抗体。真核重组质粒pcDNA3.1(+)-2019-nCoV-E经鉴定后提取质粒,免疫家兔获抗E多克隆抗体,通过Western blot和间接ELISA测定多克隆抗体特异性及效价。结果原核、真核重组质粒鉴定正确,重组E蛋白以包涵体形式表达分子质量单位约为53 ku;ELISA法测定抗E多克隆抗体效价分别达1∶16000和1∶12800。Western blot显示,重组E蛋白能被抗SARS-CoV-2 E多克隆抗体识别。结论获得重组E蛋白及兔源抗SARS-CoV-2 E多克隆抗体,可为新冠病毒感染的血清抗体筛查、抗原蛋白检测,以及药物靶点研究、E蛋白生物学特性研究和新冠快速检测试剂的研发提供支持。Objective To prepare a 2019-nCoV envelope protein and polyclonal antibodies against that protein.Methods After identification and verification,the prokaryotic plasmid pMAT9 s-2019-nCoV-E was transformed into BL21 competent cells,and expression of the recombinant envelope protein was induced with IPTG.The recombinant envelope protein was purified using gradient urea renaturation and Ni;affinity chromatography.Anti-envelope protein polyclonal antibodies were obtained by immunizing rabbits.The eukaryotic recombinant plasmid pcDNA3.1(+)-2019-nCoV-E was extracted after identification,and polyclonal antibodies against the 2019-nCoV envelope protein were obtained by immunizing rabbits.The specificity and titer of polyclonal antibodies against the 2019-nCoV-E protein were determined using Western blotting and indirect ELISA.Results The prokaryotic and eukaryotic recombinant plasmids were identified as correct.The molecular weight of the recombinant envelope protein expressed in inclusion bodies was about 53 ku.The titers of anti-envelope protein polyclonal antibodies were determined to be re 1:16000 and 1:12800,respectively,according to ELISA.The recombinant envelope protein was recognized by anti-SARS-CoV-2 envelope protein polyclonal antibodies according to Western blotting.Conclusion A recombinant envelope protein and rabbit anti-SARS-CoV-2 envelope protein polyclonal antibodies were obtained.These can be used to screen serum antibodies,detect antigen proteins,research drug targets,and support research into the biological characteristics of the envelope protein and rapid detection reagents to detect SARS-CoV-2.

关 键 词：SARS-CoV-2 E 原核表达 多克隆抗体 E蛋白 

分 类 号：R373.1[医药卫生—病原生物学]