摘　要：新型冠状病毒严重急性呼吸综合征冠状病毒2（SARS-CoV-2）感染引发的新型冠状病毒肺炎（COVID-19）疫情在全球持续流行，疫苗的研发和推广使用是阻止新冠疫情的关键手段。SARS-CoV-2核衣壳蛋白（NP）作为病毒的主要结构蛋白，是疫苗开发的潜在候选靶点。鞭毛素B（FlaB）可作为免疫佐剂，增强抗原的免疫原性。本研究对NP和NP-FlaB融合蛋白的免疫原性开展了研究，利用大肠杆菌表达系统分别表达纯化了NP、NP-FlaB融合蛋白，将抗原通过皮下或鼻内途径免疫BALB/c小鼠，分析血清中NP特异性免疫球蛋白G（IgG）、黏膜中NP特异性免疫球蛋白A（IgA）和NP特异性细胞因子分泌的T细胞应答。结果表明：一次皮下免疫NP或NP-FlaB融合蛋白足以引起抗NP的血清IgG抗体反应，能有效诱导分泌白细胞介素4（IL-4）的NP特异性效应T细胞，但NP和NP-FlaB融合蛋白组别之间无显著性差异；鼻内途径免疫下，NP-FlaB融合蛋白免疫组血清中NP特异性IgG抗体滴度和肺内黏膜IgA抗体滴度显著高于NP组。整体结果显示，SARS-CoV-2 NP和NP-FlaB融合蛋白具有很强的免疫原性，NP-FlaB融合蛋白能引起黏膜免疫应答，两者均可作为SARS-CoV-2疫苗的候选蛋白。SARS-CoV-2 NP及NP-FlaB融合蛋白的免疫原性的探究为后续新冠病毒NP疫苗开发提供了新的思路和参考。
关键词：NP；SARS-CoV-2；免疫原性；黏膜免疫；亚单位疫苗
中图分类号：R563.1；R181.3　　　　　文献标志码：ADOI：10.3969/j.issn.1007-7146.2021.04.002

Abstract: The COVID-19 pandemic caused by SARS-CoV-2 is still prevalent around the world. Vaccine development, promotion, usage are key means to prevent COVID-19. Nuclearcapsid protein (NP), as the main structural protein of SARS-CoV-2，is a potential candidate target for vaccine development. Flagellin B (FlaB) has been used as an immune adjuvant to enhance the immunogenicity of antigens. In this research, we analyzed the immunogenicity of NP and a NP-FlaB fusion protein. Firstly, the NP, NP-FlaB fusion protein were expressed and purified by the E. coli system; then the BALB/c mice were immunized with the antigens by subcutaneous or intranasal route; the NP-specific serum IgG, mucosal IgA and cytokine secreting T cell responses were analyzed. The results showed that: one-time subcutaneous immunization of NP or NP-FlaB fusion protein was sufficient to elicit robust anti-NP serum IgG antibody response and IL-4 secreting T cell response, and no significant differences were observed between NP and NP-FlaB fusion protein. However, when inoculated by the intranasal route, NP-FlaB fusion protein introduced significantly higher NP-specific serum IgG titer, as well as the higher mucosal IgA titer at the resident of lung. The overall results showed that, SARS-CoV-2 NP and NP-FlaB fusion protein are highly immunogenicity, NP-FlaB fusion protein can induce mucosal immune response, both of them are effective candidates for SARS-CoV-2 vaccine. In summary, our research on the immunogenicity of NP and NP-FlaB of SARS-CoV-2 provided a new idea and reference for the subsequent development of SARS-CoV-2 vaccine based on NP.
Key words: NP; SARS-CoV-2; immunogenicity; mucosal immunity; subunit vaccine
（Acta Laser Biology Sinica, 2021, 30(4): 296-304）