两种表达系统来源新冠病毒RBD蛋白免疫小鼠后的IgG抗体水平对比研究  

Comparison of IgG Antibody Levels in Mice Immunized with SARS-CoV-2 RBD Proteins Produced from Two Expression Systems

作　　者：胡立强[1,2] 苏涛 张勇[1] 杨浩[1] 陶怡然 陶泽 HU Li-Qiang;SU Tao;ZHANG Yong;YANG Hao;TAO Yi-Ran;TAO Ze(Key Laboratory of Transplant Engineering and Immunology,MOH,West China Hospital,Sichuan University,Chengdu 610041,China;West China-California Research Center for Predictive Intervention Medicine,West China Hospital,Sichuan University,Chengdu 610041,China)

机构地区：[1]四川大学华西医院卫健委移植工程与移植免疫重点实验室,成都610041 [2]四川大学华西医院华西-加州预测干预医学研究中心,成都610041

出　　处：《中国生物化学与分子生物学报》2022年第8期1053-1061,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.82172731,82003643)资助。

摘　　要：当前新冠肺炎疫情仍在全球蔓延,给中国及世界的公共卫生安全和经济发展带来了严峻挑战。SARS-CoV-2病毒入侵机体的关键过程是刺突蛋白受体结合域RBD通过结合宿主细胞ACE2受体实现感染,而此过程与病原快速检测、疫苗以及药物干预等主要抗病毒策略的研究与开发密切相关。因此,本研究旨在比较哺乳细胞和昆虫细胞重组表达来源的新冠病毒刺突蛋白受体结合结构域RBD免疫小鼠后产生IgG抗体水平的变化,评估不同来源和不同剂量抗原的抗体滴度水平和持续时间,希望将有助于疫苗、药物以及病原检测等相关研究。通过SDS-PAGE和质谱技术鉴定出昆虫和哺乳动物细胞表达系统制备的bRBD和hRBD蛋白质的分子量略有差异,主要为糖基化修饰不同所致;流式细胞术检测发现,bRBD和hRBD与ACE2受体过表达细胞结合率分别为88.5%和92.7%,表明二者均具有较好的活性;利用Quick Antibody免疫佐剂通过肌肉注射对Balb/c小鼠进行免疫接种,设置10μg、20μg 2个免疫抗原的剂量,共接种2次,并在初次免疫后第2、4、6、8、12、24周从尾部取血,分离获得血清;酶联免疫吸附结果显示,10μg和20μg的bRBD和hRBD蛋白质免疫均能够快速引起小鼠的免疫反应产生IgG抗体,4~6周抗体滴度达到峰值;2种抗原均具有良好的免疫持久性。2种抗原及2个免疫剂量诱导小鼠产生的抗体滴度无明显差异;血清抗体中和试验发现,bRBD和hRBD蛋白质免疫小鼠后产生的特异性抗体,均能够抑制SARS-COV-2 Spike假病毒对ACE2阳性细胞的感染。这些结果表明,昆虫细胞和哺乳动物细胞表达系统生产的RBD蛋白,通过良好的佐剂接种均能快速引起机体有效的体液免疫应答,产生特异性中和抗体,有望用于各种RBD突变体抗体的制备。The COVID-19 pandemic is still spreading around the world,posing a severe challenge to public health security and economic development in China and beyond.The pivotal process of SARS-COV-2 invasion is that the receptor binding domain(RBD)of Coronavirus spike protein binds with the angiotensin converting enzyme 2(ACE2)receptor on host cells.Therefore,RBD is closely related to our major anti-epidemic strategies such as rapid pathogen detection and development of vaccines and antiviral drugs.In this study,we aim to compare the anti-RBD IgG by immunizing mice with the recombinant RBDs of novel Coronavirus spike protein,and evaluate the antibody titers and duration elicited by RBD produced from different host cells and used in different doses,which can be used for vaccine evaluation,drug development,and pathogen detection.SDS-PAGE and mass spectrometry analysis showed that the molecular weight of bRBD(produced by insect cells)and hRBD(produced by mammalian cells)were slightly different,which are mainly caused by different glycosylation modification.Flow cytometry revealed that the binding rates of bRBD and hRBD to HEK293-ACE2-overexpressing cells were 88.5%and 92.7%,respectively,indicating that both antigens have favorable bioactivity.Balb/c mice are immunized intramuscularly with 10μg and 20μg per mouse of RBD proteins,which were mixed with Quick Antibody adjuvant previously,and blood samples collected from the tail at 2,4,6,8,12 and 24 weeks after the primary immunization.Enzyme-linked immunosorbent assay(ELISA)showed that both RBD immunization with 10μg or 20μg could induce a rapid immune response to produce IgG antibodies.The antibody titers reached the peak at 4-6 weeks post first immunization,and persisted over a long time up to 6 months.No significant difference was observed in the induced antibody titers by bRBD and hRBD as well as their different doses.Virus Neutralization Assays showed that specific antibodies induced by RBD-immunized mice could inhibit the infection of ACE2-positive cells by SARS-CoV-2

关 键 词：新型冠状病毒 刺突蛋白 受体结合区蛋白 抗体 疫苗 

分 类 号：Q71[生物学—分子生物学]