仙台病毒递送新型冠状病毒RBD蛋白鼻内免疫接种增强呼吸道黏膜和系统性免疫应答的初步研究  

Preliminary study on intranasal immunization of SARS-CoV-2 RBD protein delivered by Sendai virus to enhance respiratory mucosal and systemic immune response

作　　者：袁明城 张剑 陈洋 薛潘 武福星 袁若栋 董阳超 徐志凯 张芳琳 雷迎峰 YUAN Mingcheng;ZHANG Jian;CHEN Yang;XUE Pan;WU Fuxing;YUAN Ruodong;DONG Yangchao;XU Zhikai;ZHANG Fanglin;LEI Yingfeng(Department of Microbiology and Pathogen Biology,School of Basic Medical Sciences,Air Force Medical University,Xi'an 710032,China)

机构地区：[1]空军军医大学基础医学院微生物与病原生物学教研室,陕西西安710032

出　　处：《空军军医大学学报》2023年第7期609-614,共6页Journal of Air Force Medical University

基　　金：国家自然科学基金(81871697);陕西省重点研发计划项目(2021ZDLSF01-05)。

摘　　要：目的 研究仙台病毒(SeV)作为递送载体在增强新型冠状病毒(SARS-CoV-2)RBD蛋白诱导呼吸道黏膜和系统性免疫应答的特征。方法 利用同源重组技术构建SARS-CoV-2 RBD与抗体Fc融合蛋白编码基因的真核表达质粒,将其转染293-T细胞中进行表达并用Western blotting验证;将RBD-Fc融合蛋白表达质粒转染293-T细胞大量表达并使用亲和层析进行蛋白纯化;通过SeV递送RBD-Fc蛋白进行鼻内免疫接种,同时设置RBD-Fc组、SeV组、PBS组作为对照,各组分别在2周后加强免疫1次,3周后检测血清样本和肺泡灌洗液(BALF)中特异性抗体水平。结果 RBD、Fc蛋白编码基因片段通过重叠PCR方法得到融合蛋白基因片段(RBD-Fc),成功构建pCAGGS-RBD-Fc真核表达质粒;Western blotting结果显示RBD-Fc融合蛋白的表达条带符合预期大小,大量表达后经亲和层析获得一条单一的接近Mr 60 000的条带,与预期大小一致,纯度高;与RBD-Fc组、SeV组、PBS组相比,SeV递送RBD-Fc蛋白通过鼻腔加强免疫小鼠后的血清样本和BALF中均可检测到RBD特异性IgG(血清样本:P<0.01;BALF样本:P<0.05),且BALF中RBD特异性IgA相比RBD-Fc组有一定升高。结论 SeV在递送SARS-CoV-2 RBD蛋白进行鼻内免疫接种方面能够诱导一定水平的黏膜免疫和系统性免疫,为呼吸道传染病的黏膜疫苗研究奠定了初步的实验基础。Objective To study the characteristics of Sendai virus(SeV)as a delivery vector in enhancing the respiratory mucosal and s ystemic immune response induced by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)RBD protein.Methods The eukaryotic expression plasmid of SARS-CoV-2 RBD and antibody Fc fusion protein-coding gene was constructed by homologous recombination technology,and transfected into 293-T cells for expression and verified by Western blotting.The expression plasmid of RBD-Fc fusion protein was transfected into 293-T cells for large-scale expression and was purified by affinity chromatography.RBD-Fc protein was delivered by SeV for intranasal immunization,and RBD-Fc group,SeV group and PBS group were set as controls.Each group was given booster immunization once after 2 weeks,and the levels of specific antibodies in serum samples and bronchoalveolar lavage fluid(BALF)were detected after 3 weeks.Results The pCAGGS-RBD-Fc eukaryotic expression plasmid was successfully constructed by overlapping PCR to obtain the fusion protein gene fragment(RBD-Fc)of RBD and Fc protein-encoding gene fragments.Western blotting results showed that the expression band of RBD-Fc fusion protein was in line with the expected size.After large-scale expression,a single band close to M r 60000 was obtained by affinity chromatography,which was consistent with the expected size and high purity.Compared with RBD-Fc group,SeV group and PBS group,RBD-specific IgG could be detected in serum samples and BALF of mice immunized by SeV delivering RBD-Fc protein through nasal cavity(serum:P<0.01,BALF:P<0.05),and RBD-specific IgA in BALF was higher than that in RBD-Fc group.Conclusion SeV can induce a certain level of mucosal and systemic immunity in delivering SARS-CoV-2 RBD protein for intranasal immunization,providing a preliminary experimental basis for the study of mucosal vaccines for respiratory infectious diseases.

关 键 词：新型冠状病毒 RBD-Fc蛋白 黏膜免疫 仙台病毒 

分 类 号：R392[医药卫生—免疫学]