展示口蹄疫病毒优势中和表位的铁蛋白纳米颗粒制备及其在小鼠模型上的免疫原性评价  

Preparation of Ferritin Nanoparticles Displaying the Dominant Neutralizing Epitopes of Foot-and-mouth disease virus and Their Immunogenicity Evaluation in Mouse(Mus musculus)Models

作　　者：卢炳州 茹毅 郝荣增 李亚军 杨洋[2] 杨锐[1] 毛玉涵 蒋成辉 陶世宇 郑海学[2] 余四九[1] 崔燕[1] LU Bing-Zhou;RU Yi;HAO Rong-Zeng;LI Ya-Jun;YANG Yang;YANG Rui;MAO Yu-Han;JIANG Cheng-Hui;TAO Shi-Yu;ZHENG Hai-Xue;YU Si-Jiu;CUI Yan(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences/State Key Laboratory of Veterinary Etiological Biology,Lanzhou 730046,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]中国农业科学院兰州兽医研究所/家畜疫病病原生物学国家重点实验室,兰州730046

出　　处：《农业生物技术学报》2024年第5期1216-1226,共11页Journal of Agricultural Biotechnology

基　　金：甘肃省重点人才项目(2022RCXM016);中国农业科学院兰州兽医研究所所级基本科研业务费(1610312021013,1610312022009)。

摘　　要：蛋白纳米颗粒能够在表面展现多拷贝抗原,模拟病毒的结构形态,更有利于免疫细胞摄取和促进抗原递呈,从而增强疫苗保护效力。本研究旨在利用大肠杆菌(Escherichia coli)表达系统制备展示口蹄疫病毒(Foot-and-mouth disease virus,FMDV)优势中和表位的铁蛋白纳米颗粒,并评估其免疫原性。首先以幽门螺杆菌(Helicobacter pylori)铁蛋白序列为基础,将FMDV优势中和表位序列插入铁蛋白第33和34位氨基酸之间,同时将耶尔森氏菌属(Yersinia)T细胞表位序列插入铁蛋白氨基酸序列的氨基末端,构建具有铁蛋白序列、B细胞表位(优势中和表位)和T细胞表位的重组表达质粒pET28a-FBT,以仅插入铁蛋白序列的重组质粒pET28a-Fer作为对照。将重组质粒转入大肠杆菌感受态细胞,经诱导表达和分子筛层析纯化获得目的蛋白,利用SDS-PAGE、Western blot、粒径分析和透射电镜进行鉴定,进一步免疫BALB/c小鼠(Mus musculus)评价免疫原性。结果显示,利用大肠杆菌表达系统成功制备的铁蛋白纳米颗粒FBT可与FMDV阳性血清发生特异性反应;FBT免疫14 d即产生血清抗体,并持续升高,在免疫35 d中和抗体效价最高可达1∶128;同时,FBT免疫小鼠的脾淋巴细胞增殖水平和白介素2(interleukin 2,IL-2)、IL-4、IL-10、干扰素-γ(interferon-γ,IFN-γ)分泌水平显著高于对照组(P<0.05)。本研究制备了展示FMDV中和表位且具有良好免疫原性的铁蛋白纳米颗粒,为新型口蹄疫基因工程疫苗的研制提供技术支持。Nanoparticles(NPs)can display multiple copies of antigen on the surface and simulate the structural morphology of viruses,which is more conducive to uptake by immune cells and promote antigen presentation,thus enhancing protective efficacy.This study aimed to generate ferritin nanoparticles harboring a dominant neutralizing epitope of Foot-and-mouth disease virus(FMDV)by using the Escherichia coli expression system,and to evaluate its immunogenicity.The recombinant expression plasmid pET28a-FBT was constructed based on the Helicobacter pylori ferritin,the dominant neutralizing epitope sequence of FMDV was inserted between the 33th and 34th amino acids of ferritin,and the T cell epitope sequence was inserted at the N-terminus of ferritin.The recombinant expression plasmid pET28a-Fer expressing Fer protein was used as control.The recombinant plasmid pET28a-Fer and pET28a-FBT were transformed into Escherichia coli competent cells,the target proteins were obtained by induced expression and size exclusion chromatography purification,and identified using SDS-PAGE,Western blot,particle size analysis,and transmission electron microscopy,then evaluated immunogenicity by immunizing BALB/c mice(Mus muculus).The results showed that the recombinant protein Fer and FBT were successfully prepared by using prokaryotic expression system.The recombinant protein FBT could be recognized specifically by FMDV positive serum.After 14 d of immunization with FBT,serum antibodies were produced and continued to rise,and the neutralizing antibody titer could reach up to 1∶128 on 35 d.At the same time,the proliferation level of spleen lymphocytes and the secretion level of interleukin 2(IL-2),IL-4,IL-10,and interferon-γ(IFN-γ)in FBT immunized mice were significantly higher than the control(P<0.05).This study prepared ferritin nanoparticles displaying FMDV neutralizing epitopes with good immunogenicity,providing technical support for the development of a novel foot-and-mouth disease engineered vaccine.

关 键 词：铁蛋白纳米颗粒 口蹄疫病毒(FMDV) 中和表位 T细胞表位 免疫原性 

分 类 号：S855.3[农业科学—临床兽医学]