基于鱼精蛋白递送SARS-CoV2 DNA-疫苗的制备及体外表达与体内免疫分析  

Preparation of SARS-CoV-2 DNA vaccine based on protamine delivery,in vitro expression and in vivo immunoassay

作　　者：刘瑛琪 李静如 孟繁 邢昊楠 郑爱萍 LIU Yingqi;LI Jingru;MENG Fan;XING Haonan;ZHENG Aiping(Instituteof Toricologyand Pharmacology,Academy of Military Medical Sciences,Beijing 100850,China;North China University of Science and Technology)

机构地区：[1]中国人民解放军军事科学院军事医学研究院毒物药物研究所,北京100850 [2]华北理工大学药学院

出　　处：《中国病原生物学杂志》2023年第9期993-999,共7页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.82104105)。

摘　　要：目的 利用非病毒基因载体的鱼精蛋白递送SARS-CoV-2 DNA,筛选最佳比例的PS-RBD纳米粒,并对其进行体外摄取能力与体内免疫水平评价。方法 选择优化的RBD基因序列构建重组RBD质粒,不同量的鱼精蛋白与pDNA经过室温孵育可得不同N/P比PS-RBD纳米粒。马尔文粒径仪测定粒径、PDI及Zeta电位,透射电镜观察形态。核酸凝胶电泳和结合沉淀试验检测鱼精蛋白保护pDNA抗剧烈外力和核酸酶的降解情况,CCK8法检测其细胞毒性,激光共聚焦显微镜和流式细胞检测比较其与裸DNA对DC2.4细胞转染率。用PS-RBD疫苗免疫小鼠,检测小鼠血清中IgG抗体和IFN-γ水平。结果 5/1的PS-RBD纳米粒形态规整,平均粒径为(235.0±4.7)nm,其保护pDNA抵抗剧烈外力和核酸酶的降解效果最佳,稳定性良好,细胞毒性低,安全性高。体外转染试验显示SARS-CoV-2 RBD基因可进入细胞并表达,体内免疫试验表明该疫苗可诱导小鼠产生体液免疫及细胞免疫应答。结论 鱼精蛋白作为非病毒基因载体制备工艺简单、性能良好,用PS-RBD疫苗免疫小鼠可诱导产生体液免疫及细胞免疫应答,可为SARS-CoV-2疫苗研发提供新思路。Objective This study aimed to deliver SARS-CoV-2 DNA using a non-viral gene carrier,protamines,to identify the optimal proportion of PS-RBD nanoparticles,and evaluate their in vitro uptake capability as well as in vivo immunogenicity.Methods An optimized RBD gene sequence was selected to construct the recombinant RBD plasmid.Different quantities of protamines and pDNA were incubated at room temperature to obtain PS-RBD nanoparticles at varying N/P ratios.The Malvern particle size analyzer was used to measure the size,PDI,and Zeta potential of the nanoparticles,while their morphology was observed using a transmission electron microscope.Gel electrophoresis and binding precipitation tests were employed to examine the protection offered by protamines to pDNA against external forces and nucleic acid enzyme degradation.The CCK8 assay was used to assess cytotoxicity,and laser confocal microscopy and flow cytometry were used to compare the transfection rates of the nanoparticles and naked DNA into DC2.4 cells.Mice were im*munized with the PS-RBD vaccine,and their serum IgG antibody and IFN-levels were measured.Results PS-RBD nanoparticles at a 5/1 ratio exhibited uniform morphology with an average particle size of(235.0±4.7)nm.These nanoparticles displayed the best protective effects against external forces and nucleic acid enzyme degradation,demonstrating good stability,low cytotoxicity,and high safety.In vitro transfection experiments revealed that the SARS-CoV-2 RBD gene could enter cells and express with a positive transfection rate of up to 34.5%.In vivo immunological tests showed that after three immunizations with the PS-RBD vaccine,the IgG antibody titer reached 1o°,with significant differences in IgG and IFN-levels compared to the naked DNA group.The vaccine was found to induce both humoral and cellular immune responses in mice.Conclusion protamines,as non-viral gene carrier,offers a simple preparation process and excellent performance.Immunizing mice with the PS-RBD vaccine can induce humoral and cellular im

关 键 词：鱼精蛋白 SARS-CoV-2 DNA疫苗 体内免疫 

分 类 号：R373.1[医药卫生—病原生物学]