目的: 采用新冠病毒S蛋白受体结合区域（receptor binding region of the S protein of COVID-19,RBD）mRNA-LNP为免疫原的新型免疫策略,制备RBD鼠源单克隆抗体（monoclonal antibody,McAb）,并进行相关的特异性鉴定。方法: 该研究采用含有编码RBD蛋白序列的mRNA-LNP作为免疫原,采用肌肉注射RBD mRNA-LNP免疫Balb/c小鼠并通过流式细胞术（flow cytometry,FCM）、酶联免疫吸附测定（enzyme-linked immunosorbent assay,ELISA）检测小鼠血清中RBD 抗体生成滴度;血清滴度检测达标后利用杂交瘤融合细胞技术构建稳定分泌RBD McAb 的杂交瘤细胞、腹水生产及抗体纯化;用免疫荧光（immunofluorescence,IF）、FCM、ELISA、免疫印迹（Western-blot）等方法鉴定其生物活性,检测所得抗体的特异性。结果: 复筛后获得两株能稳定分泌RBD McAb的杂交瘤细胞,分别命名为2D7D11、9B6H5。经检测2D7D11、9B6H5的Ig 亚类均为 IgG2b,轻链为κ链;经IF、FCM、ELISA和Western-blot检测证实：2D7D11、9B6H5两株 RBD McAb 与 RBD mRNA-LNP转染细胞表达的RBD蛋白或S-RBD（e1）-PET28重组蛋白均具有较高的特异性。结论: 本研究成功制备了2D7D11、9B6H5两株抗RBD特异性的McAb,为mRNA-LNP作为新型免疫原在动物体内的递送、翻译与修饰表达、自主引起体液免疫反应从而产生抗体以及相关的检测方法开发、功能探究等提供了试验基础;为研究因各种原因无法制备有效的免疫原而受到阻碍的抗体开发项目,提供了一种有效的新型免疫策略。
Abstract
Objective To prepare monoclonal antibody (McAb) against COVID-19 in mice by using mRNA-LNP, the receptor binding region of the S protein of COVID-19 (RBD), as the immunogen. Methods This study used mRNA LNP containing the RBD protein sequence as an immunogen, . Immunization of Balb/c mice with RBD mRNA LNP via intramuscular injection. The RBD antibody titer in the serum of the mice was detected by flow cytometry (FCM) and enzyme-linked immunosorbent assay (ELISA) ; After achieving the standard for serum titer detection, stable hybridoma cells secreting RBD monoclonal antibodies were constructed using hybridoma fusion cell technology, followed by ascites production and antibody purification; Use immunofluorescence (IF), flow cytometry (FCM), enzyme-linked immunosorbent assay (ELISA), Western blot and other methods to identify its biological activity and detect the specificity of the obtained antibodies. Result After re screening, two hybridoma cells capable of stably secreting RBD McAbs were obtained and named 2D7D11 and 9B6H5, respectively. After testing, the Ig subclasses of 2D7D11 and 9B6H5 are both IgG2b, with the light chain being the kappa chain; Through IF, FCM, ELISA, and Western blot analysis, it was confirmed that the 2D7D11 and 9B6H5 RBD McAbs exhibit high specificity towards RBD protein or S-RBD (e1) - PET28 recombinant protein expressed in RBD mRNA LNP transfected cells. Conclusion This study successfully prepared two anti RBD specific monoclonal antibodies, 2D7D11 and 9B6H5, This provides an experimental basis for the delivery, translation, and modified expression of mRNA LNP as a novel immunogen in animals, the autonomous generation of humoral immune responses to produce antibodies, and the development of related detection methods and functional exploration; This provides an effective novel immune strategy for antibody development projects that are hindered by the inability to prepare effective immunogens due to various reasons.
关键词
新冠病毒S蛋白受体结合区域 /
mRNA-LNP /
免疫原 /
免疫策略 /
单克隆抗体
Key words
receptor binding region of the S protein of COVID-19 (RBD) /
mRNA-LNP /
immunogen /
immune strategy /
monoclonal antibody
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基金
湖南省自然科学基金部门联合基金项目“基于mRNA-LNP技术开发Epcam-CD3双抗用于结直肠癌治疗的基础研究”（2024JJ8229）;湖南省重点研发计划项目“应用人源结直肠癌动物模型研发和评价新型免疫治疗药物”（2024DK2002）
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