猫疱疹病毒多表位纳米颗粒疫苗的制备及免疫原性评价

doi:10.16431/j.cnki.1671-7236.2025.09.024

摘要/Abstract

摘要： 【目的】设计并开发针对猫疱疹病毒1型(FHV-1)gB、gD蛋白的多表位纳米颗粒疫苗，为FHV-1多表位纳米颗粒疫苗的抗原筛选与优化提供理论依据。【方法】本研究利用多种生物信息学工具对 FHV-1 gB、gD蛋白的理化性质、跨膜结构、N-糖基化位点、二级结构、B细胞抗原表位、免疫原性和保守性进行分析。构建重组质粒pET20b-FBDF,转化大肠杆菌BL21(DE3)感受态细胞进行诱导表达。经 Ni柱亲和层析纯化及透析浓缩处理后得到重组蛋白FBDF，通过 SDS-PAGE 进行鉴定，BCA 法测定蛋白含量，透射电子显微镜(TEM)和动态光散射(DLS)观察自组装纳米颗粒的形态和粒径大小。将制备的FHV-1表位纳米颗粒疫苗皮下免疫 BALB/c 小鼠，评价其免疫原性。【结果】 FHV-1 gB、gD蛋白的多个氨基酸域均表现出优异的亲水性、抗原性、结构柔韧性和表面可及性特征。FHV-1 gB、gD蛋白分别存在11和5个潜在的糖基化位点；蛋白二级结构均主要由α-螺旋、β-折叠、β-转角及无规则卷曲组成，其中无规则卷曲占比最高；分别产生17和6个B细胞线性表位。筛选出3个高抗原性和保守性的优势抗原表位：gB₁：197－208，gB₂:314－325，gD: 221－232。将表位使用柔性连接子(GGGGS)与 Ferritin 相连，经密码子优化后，构建 pET20b-FBDF重组质粒。SDS-PAGE结果显示，重组蛋白 FBDF 在 30 ℃、0.5 mmol/L IPTG 诱导16 h时成功可溶性表达。TEM和DLS结果显示，纯化后的 FBDF 蛋白可自组装成直径为16.55 nm 的纳米颗粒。小鼠免疫结果显示，三免后14 d小鼠血清中抗体滴度达到峰值，其中 FBDF＋Al(OH)₃、FBDF＋CpG、FRCPV组抗体滴度分别为1∶680 000、1∶15 600、1∶3 500，显著高于PBS和 Ferritin 组(P＜0.05)。接种FBDF可诱导小鼠产生较高水平的γ-干扰素(IFN-γ)、白细胞介素-4(IL-4)和中和抗体，中和抗体水平最高可达 1∶2⁵。【结论】本研究构建的多表位纳米颗粒疫苗FBDF具有较好的免疫原性，可诱导BALB/c小鼠产生体液免疫和细胞免疫应答，为 FHV-1 多表位疫苗的研究奠定基础。

关键词: 猫疱疹病毒(FHV); gB蛋白; gD蛋白; 抗原表位预测; 多表位疫苗

Abstract: 【Objective】 This study aimed to design and develop the multi-epitope nanoparticle vaccine against gB and gD proteins of Feline herpesvirus type 1 (FHV-1), and provide a theoretical basis for antigen screening and optimization of FHV-1 multi-epitope nanoparticle vaccines. 【Method】 In this study, a variety of bioinformatics tools were used to analyze the physicochemical properties, transmembrane structure, N-glycosylation site, secondary structure, B cell epitope, immunogenicity and conservation of FHV-1 gB and gD proteins. The recombinant plasmid pET20b-FBDF was constructed and transformed into Escherichia coli BL21 (DE3) competent cells to induce expression. The recombinant protein FBDF were purified by Ni column affinity chromatography and dialyed concentration treatment, and then identified by SDS-PAGE, the protein content was determined by BCA. The morphology and particle size of self-assembled nanoparticles were observed by transmission electron microscope (TEM) and dynamic light scattering (DLS). The prepared immunogens were immunized subcutaneously in BALB/c mice to evaluate their immunogenicity. 【Result】 Multiple amino acid domains in FHV-1 gB and gD proteins exhibited excellent hydrophilicity, antigenicity, structural flexibility, and surface accessibility characteristics. FHV-1 gB and gD proteins had 11 and 5 potential glycosylation sites, respectively. The secondary structures of FHV-1 gB and gD proteins were primarily composed of alpha helix, beta sheet, beta turns, and random coil, with random coil constituting the predominant structural component. And FHV-1 gB and gD proteins produced 17 and 6 B-cell linear epitopes, respectively. Three dominant antigenic epitopes with high antigenicity and conservation were screend， which were gB₁: 197－208, gB₂: 314－325 and gD: 221－232, respectively. The epitope was connected with Ferritin by flexible linker (GGGGS). After codon optimization, the recombinant plasmid pET20b-FBDF was constructed. SDS-PAGE results showed that the recombinant protein FBDF was successfully expressed at 30 ℃, 0.5 mmol/L IPTG for 16 h. The results of TEM and DLS showed that the purified FBDF protein could be self-assembled into nanoparticles with a diameter of 16.55 nm. The results of mouse immunization showed that serum antibody titers peaked after 14 days of triple immunization, and the antibody titers in FBDF+Al(OH)₃, FBDF+CpG and FRCPV groups were 1∶680 000, 1∶15 600 and 1∶3 500, respectively, which were significantly higher than those in PBS and Ferritin groups (P＜0.05). Vaccination with FBDF could induce mice to produce higher levels of interferon-γ (IFN-γ), interleukin-4 (IL-4), and neutralizing antibodies, with neutralizing antibody levels up to 1∶2⁵. 【Conclusion】 The multi-epitope nanoparticle vaccine FBDF constructed in this study had good immunogenicity and could induce humoral and cellular immune responses in BALB/c mice， which laid a foundation for the research of FHV-1 multi-epitope vaccine.

Key words: Feline herpesvirus (FHV); gB protein; gD protein; antigen epitope prediction; multi-epitope vaccine

中图分类号:

S852.65

杨丹丹, 刘冰, 赵维昊, 赵亚楠, 张玉蝶, 郭巾玲, 刘冬禹, 殷玉和, 吴丛梅. 猫疱疹病毒多表位纳米颗粒疫苗的制备及免疫原性评价[J]. 中国畜牧兽医, 2025, 52(9): 4260-4272.

YANG Dandan, LIU Bing, ZHAO Weihao, ZHAO Yanan, ZHANG Yudie, GUO Jinling, LIU Dongyu, YIN Yuhe, WU Congmei. Preparation and Immunogenic Evaluation of Multi-epitope Nanoparticles Vaccine of Feline Herpesvirus[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(9): 4260-4272.

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