针对SARS-CoV-2核衣壳蛋白的单克隆抗体的制备和鉴定

Preparation and identification of monoclonal antibodies against the nucleocapsid protein of SARS-CoV-2

目的研制SARS-CoV-2的N蛋白单克隆抗体,并对其特异性识别真核表达N蛋白等特性进行鉴定,为下一步应用奠定材料基础。方法利用大肠杆菌表达并纯化SARS-CoV-2重组N蛋白,制备并筛选分泌N蛋白特异性单克隆抗体的阳性B细胞杂交瘤,制备腹水后利用间接ELISA法测定单抗的效价、亚型和亲和力;通过Western blot、间接免疫荧光试验分析其与原核、真核表达SARS-CoV-2重组N蛋白的特异性结合能力;通过间接ELISA法评价其在不同冠状病毒N蛋白检测中的应用潜力。结果成功地表达和纯化SARS-CoV-2重组N蛋白,筛选获得1株特异性识别重组N蛋白的单克隆抗体并命名为11F4,其亚型为IgG1,腹水效价为2.0×10^(7)以上,亲和力为4.44×10^(8)M^(-1);Western Blot分析表明其能与SARS-CoV-2重组N蛋白特异性反应,间接免疫荧光试验显示其能特异性识别HEK-293T细胞表达的SARS-CoV-2 N蛋白,表明该蛋白免疫反应性良好;间接ELISA法结果表明其可特异性识别真核细胞表达的SARS-CoV-2 N蛋白,而与MERS-CoV和HCoV-229E的N蛋白无交叉反应。结论成功获得可特异性识别细胞表达及体外制备的SARS-CoV-2 N蛋白的单克隆抗体,具备特异性检测SARS-CoV-2的潜能,为下一步研究提供了重要生物材料。

In this study,monoclonal antibodies(mAb)agai nst SARS-CoV-2 N protein were developed,and their specific recognition of eukaryotically expressed SARS-CoV-2 N protein were identified.The recombinant N protein of SARS-CoV-2 expressed and purified from Escherichia coli was used to generate specific hybridoma cells through B cell hybridoma technology.Positive hybridoma cells that secreted mAbs specifically recognizing recombinant N protein were screened,and their biological characteristics,including subtype,titer,affinity,and specificity,were determined by Western blotting and indirect immunofluorescence assays.Indirect ELISA was used to evaluate the application potential of the mAbs in the detection of different coronavirus N proteins.The recombinant N protein of SARS-CoV-2 was successfully expressed and purified.One strain of hybridoma cells that secreted mAbs specific to the recombinant N protein was obtained and named 11F4.The mAbs produced by 11F4 showed biological characteristics including IgG1 subtype,ascites titer above 2.0×10^(7),and affinity of 4.44×10^(8)M^(-1).Importantly,Western blotting and indirect immunofluorescence assays demonstrated a specific response of 11F4 to SARS-CoV-2 N protein expressed by E.coli and HEK-293T cells,thus indicating the excellent ability of 11F4 to react with N protein of SARS-CoV-2.Indirect ELISA results demonstrated that 11F4 mAbs specifically recognized the SARS-CoV-2 N protein without cross-reacting with other coronavirus N proteins,such as MERS-CoV and HCoV-229E.Thus,mAbs with detection potential for specifically recognizing the N protein of SARS-CoV-2 were successfully generated,thereby providing important biological materials for further research.