携带猪流行性腹泻病毒抗原表位的铁蛋白纳米颗粒制备与免疫原性分析

doi:10.16431/j.cnki.1671-7236.2023.04.028

摘要/Abstract

摘要： 【目的】制备携带猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)抗原表位的铁蛋白纳米颗粒,并评价其免疫原性,为PEDV的防控提供新的候选亚单位疫苗。【方法】将PEDV纤突蛋白S的COE和S1D区域设计成串联表位S1CD并进行密码子优化和合成,构建单独表达S1CD蛋白的重组质粒pET28a-S1CD及融合表达S1CD蛋白和铁蛋白亚基的重组质粒pET28a-S1CD-Ferritin,并在大肠杆菌中进行诱导表达和纯化。使用SDS-PAGE和Western blotting方法验证S1CD、S1CD-Ferritin蛋白在大肠杆菌内是否正确表达,并通过透射电镜观察其形态结构。将纯化的S1CD-Ferritin蛋白免疫小鼠,同时以纯化的S1CD蛋白和PBS为对照,采用间接ELISA方法检测各组小鼠血清的S特异性抗体水平和γ-干扰素(IFN-γ)含量,通过中和试验测定中和抗体水平,分析重组蛋白在小鼠体内的免疫原性。【结果】成功构建了重组质粒pET28a-S1CD和pET28a-S1CD-Ferritin。SDS-PAGE结果显示,高效表达出了S1CD和S1CD-Ferritin重组蛋白,大小分别约为22和39 ku,均以包涵体形式存在。Western blotting鉴定结果显示,S1CD和S1CD-Ferritin蛋白均与PEDV阳性血清发生特异性反应,表明原核表达得到的S1CD和S1CD-Ferritin重组蛋白具备良好的反应原性。透射电镜结果显示,纯化后的S1CD-Ferritin蛋白可形成大小均一、粒径为12~20 nm的球形颗粒,表明S1CD-Ferritin蛋白已成功组装为纳米颗粒。血清抗体检测结果显示,首次免疫7 d后,S1CD-Ferritin蛋白免疫组抗体水平迅速提高,显著高于S1CD蛋白免疫组(P＜0.05);首次免疫28 d后,S1CD-Ferritin蛋白免疫组的中和抗体水平及IFN-γ含量均显著高于S1CD蛋白免疫组(P＜0.05)。【结论】融合铁蛋白的PEDV抗原表位在大肠杆菌中得到高效表达,并能自组装形成纳米颗粒,显著增强目的抗原的免疫原性,为研究新的PEDV亚单位疫苗拓宽了研究思路。

关键词: 猪流行性腹泻病毒(PEDV); 铁蛋白; 纳米颗粒; 原核表达; 免疫原性

Abstract: 【Objective】 The purpose of this experiment was to prepare ferritin nanoparticles carrying the epitope of Porcine epidemic diarrhea virus (PEDV) antigen and evaluate their immunogenicity,so as to provide a new candidate subunit vaccine for the prevention and control of PEDV.【Method】 The COE and S1D regions of PEDV fibrin S were designed as the tandem epitope S1CD,and the codon was optimized and synthesized.The recombinant plasmid pET28a-S1CD expressing S1CD protein alone and the recombinant plasmid pET28a-S1CD-Ferritin fusion expression S1CD protein and ferritin subunit were constructed,and then induced in E.coli and purified.SDS-PAGE and Western blotting were used to verify the expression of S1CD and S1CD-Ferritin proteins in E.coli.The morphology and structure of S1CD and S1CD-Ferritin proteins were observed by transmission electron microscope.The mice were immunized with purified S1CD-Ferritin protein,and S1CD protein and PBS were used as controls.Indirect ELISA was used to detect the level of S-specific antibody and the content of interferon-γ (IFN-γ).The level of neutralizing antibody was determined by neutralization test,and the immunogenicity of recombinant protein in mice was analyzed.【Result】 The recombinant plasmids pET28a-S1CD and pET28a-S1CD-Ferritin were successfully constructed.The result of SDS-PAGE showed that the recombinant protein S1CD and S1CD-Ferritin were efficiently expressed,with the size of about 22 and 39 ku,respectively,and both existed in the form of inclusion bodies.Western blotting results showed that the purified proteins could react specifically with the PEDV positive serum,indicating that the recombinant proteins S1CD and S1CD-Ferritin expressed by prokaryotic expression system had good reactivity.The result of transmission electron microscope showed that the purified S1CD-Ferritin protein could self-assemble into uniform nanoparticles with the diameter of 12-20 nm,indicating that S1CD-Ferritin protein had been successfully assembled into nanoparticles.Serums antibody test results showed that 7 days after the first immunization,the titer of PEDV specific antibody in S1CD-Ferritin group was significantly higher than that in S1CD group (P＜0.05).28 days after the first immunization,the level of neutralizing antibody and the content of IFN-γ were significantly higher than that in S1CD group (P＜0.05).【Conclusion】 The PEDV epitope fused with ferritin was highly expressed in E.coli,and could self-assemble into nanoparticles,and effectively enhance the immunogenicity of the protein antigens,which broadened the research ideas for studying new PEDV subunit vaccine.

Key words: Porcine epidemic diarrhea virus (PEDV); ferritin; nanoparticles; prokaryotic expression; immunogenicity

中图分类号:

S852.4

刘家兴, 韩雪英, 张鑫茹, 邓嘉昕, 姚伦广, 刘阳坤. 携带猪流行性腹泻病毒抗原表位的铁蛋白纳米颗粒制备与免疫原性分析[J]. 中国畜牧兽医, 2023, 50(4): 1567-1574.

LIU Jiaxing, HAN Xueying, ZHANG Xinru, DENG Jiaxin, YAO Lunguang, LIU Yangkun. Preparation and Immunogenicity Analysis of Ferritin Nanoparticles Carrying Antigenic Epitopes of Porcine Epidemic Diarrhea Virus[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1567-1574.

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