新型冠状病毒S2蛋白多克隆抗体制备及生物信息学分析  

Preparation of polyclonal antibodies against the SARS-CoV-2 S2 protein and bioinformatic analysis of that protein

作　　者：徐海山 林芋利 郑皖怿 高岩 杜昆朋 付玉荣[2] 伊正君[1] XU Hai-shan;LIN Yu-li;ZHENG Wan-yi;GAO Yan;DU Kun-peng;FU Yu-rong;YI Zheng jun(College of Medical Laborutory,Weifang Medical University,Weifang,Shandong 261053.China;Basic Medical College of Weifang Medical University)

机构地区：[1]潍坊医学院医学检验学院,山东淮坊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1377-1381,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(SARS-CoV-2)S2蛋白多克隆抗体,并分析其潜在的生物学功能。方法将鉴定正确的重组子pET-32a-S2转化大肠埃希菌BL21(DE3)菌株,IPTG诱导表达目的蛋白并优化蛋白表达条件。镍柱亲和层析法纯化重组S2蛋白,免疫新西兰大白兔,制备多克隆抗体,Western blot和ELISA对抗体进行特异性及效价检测。在线预测SARS-CoV-2 S2蛋白的理化性质、糖基化位点及抗原表位,并与可感染人类的冠状病毒S2蛋白进行同源序列比对,构建进化树。结果获得重组S2蛋白及多克隆抗体;Western blot检测制备的多克隆抗体可特异结合S2蛋白,ELISA检测抗体效价达1∶12800。预测该蛋白以α螺旋为主,属于疏水性蛋白,有多个糖基化位点及多个抗原表位;预测SARS-CoV-2 S2蛋白与SARS-CoV同源性较高。结论生物信息学预测SARS-CoV-2S2蛋白为疏水性蛋白且具有抗原性,制备的抗S2多克隆抗体特异性较好,为阻断新冠病毒药物和诊断试剂的研发奠定了基础。Objective To prepare polyclonal antibodies against the novel coronavirus(SARS-CoV-2)S2 protein and to analyze its potential biological functions.Methods Identified as correct,recombinant pET-32 a-S2 was transformed into the Escherichia coli BL21(DE3)strain,and IPTG was used to induce the expression of the target protein.Conditions for protein expression were optimized.The recombinant S2 protein was purified using nickel column affinity chromatography.Polyclonal antibodies were prepared by immunizing New Zealand rabbits,which were tested for specificity and potency using Western blotting and ELISA.The physicochemical properties,glycosylation sites,and epitopes of the SARS-CoV-2 S2 protein were predicted online,and homologous sequences were compared to the human infectable coronavirus S2 protein to construct an evolutionary tree.Results The recombinant S2 protein and polyclonal antibodies were obtained.The polyclonal antibody prepared using Western blotting specifically bound to the S2 protein,and the antibody potency reached 1:12800 according to ELISA.The protein was predicted to be a hydrophobic protein mainly consisting ofα-helices with multiple glycosylation sites and multiple epitopes.The SARS-CoV-2 S2 protein was predicted to have a high degree of similarity to SARS-CoV.Conclusion Bioinformatics predicted that the SARS-CoV-2 S2 protein was hydrophobic and antigenic,and the prepared anti-S2 polyclonal antibodies had good specificity.These findings have laid the foundation for the development of drugs and diagnostic reagents to block novel coronaviruses.

关 键 词：新型冠状病毒(SARS-CoV-2) 原核表达 抗原表位 多克隆抗体 

分 类 号：R373.1[医药卫生—病原生物学]