新型冠状病毒重组抗原制备、纯化及鉴定

Preparation,purification,and identification of recombinant antigens of the novel coronavirus

目的制备新型冠状病毒(SARS-CoV-2)的刺突蛋白(S)和核衣壳蛋白(N)重组抗原。方法利用Blast程序检索目标序列SARS-CoV-2的S蛋白和N蛋白的同源序列,用SWISS-MODEL软件对目标序列进行结构建模,运用DISCO TOPE对S蛋白和N蛋白的B细胞抗原表位进行分析,选取抗原表位较多的序列进行合成;重组抗原分离纯化后制备兔抗新冠病毒蛋白多抗,用Western blot检测抗原SARS-CoV-2的S蛋白以及N蛋白免疫学活性;用ELISA法检测自制抗原与市售抗体的结合能力。结果重组质粒pET-22b-SA、pET-22b-SB、pET-22b-SC、pET-22b-NA、pET-22b-NB测序鉴定结果显示正确;Western blot结果显示,重组抗原与山羊抗兔IgG-HRP有特异性结合反应,表明具有良好的免疫学反应;ELISA法结果显示,重组抗原结合能力明显强于市售抗N蛋白抗原。结论制备的抗原与不同靶IgG和IgM结合能力强,特异性高,具有开发成检测血清SARS-CoV-2特异性IgG和IgM抗体的试剂盒的潜力。

Objective To prepare the recombinant antigens spike protein(S)and nucleocapsid protein(N)of the novel coronavirus(2019-novel coronavirus,SARS-CoV-2).Methods Homologous sequences of S and N proteins of the destination sequence were retrieved using blast.The structures of the target sequences were modeled with the swiss-model software.The B-cell antigens of S and N proteins were analyzed using DISCO TOPE.The sequences of multiple epitopes were selected for synthesis.The rabbit anti-virus protein against the SARS-CoV-2 was prepared after the separation and purification of recombinant antigen.The immunological activities of S and N proteins of SARS-CoV-2 antigen were detected using with Western blot.ELISA was used to detect the binding capacity of homemade antigen and commercial antibody.Results Sequence results of the recombined plasmids including pET-22b-SA,pET-22b-SB,pET-22b-SC,pET-22b-NA,and pET-22b-NB were identified.Western blot results showed that the recombinant antigen has specific binding responses to the goat anti-rabbit IgG-HRP,suggesting a good immune response.ELISA results showed that the binding capacity of the recombinant antigen was dramatically better than the commercial N protein antigen.Conclusion The present antigen has strong binding capability against different targeting IgG and IgM with high specificity and has potential to develop the serum detection kits for the SARS-CoV-2 specific IgG and IgM antibody.