偶联非洲猪瘟病毒p30蛋白的铁蛋白纳米颗粒制备及免疫原性评价

Preparation and immunogenicity evaluation of ferritin nanoparticles conjugated with African swine fever virus p30 protein

摘要:

本研究旨在利用铁蛋白制备携带非洲猪瘟病毒p30蛋白的纳米颗粒抗原，并对其免疫原性进行评价，为非洲猪瘟纳米疫苗研究提供实验基础。首先，将编码p30蛋白和SpyTag标签的基因序列融合后插入pCold-I载体，得到pCold-p30质粒。将编码SpyCatcher与铁蛋白的基因序列融合后插入pET-28a(+)载体得到pET-F-np质粒，并分别在大肠杆菌中诱导表达。然后，将亲和层析纯化后的p30蛋白与铁蛋白在体外偶联结合，用分子筛纯化后得到偶联p30蛋白的铁蛋白纳米颗粒F-p30，利用粒径仪、透射电镜观察形态结构。将F-p30纳米颗粒蛋白免疫小鼠，评估其体液和细胞免疫应答。结果表明，本研究成功制备出偶联非洲猪瘟病毒p30蛋白的铁蛋白纳米颗粒，颗粒结构约20 nm。F-p30在体外能被小鼠骨髓源树突状细胞(bone marrow-derived dendritic cells, BMDCs)高效摄取，与p30蛋白相比，F-p30纳米颗粒免疫小鼠诱导产生更高水平的特异性抗体和细胞因子，更有利于刺激淋巴结滤泡辅助T细胞(follicular helper T cell, T_FH)、生发中心B细胞(germinal center B cell, GCB)和脾脏CD4⁺及CD8⁺T淋巴细胞的增殖。综上，本研究成功制备了偶联非洲猪瘟病毒p30蛋白的铁蛋白纳米颗粒，能显著增强p30蛋白的免疫原性，为非洲猪瘟疫苗的研究奠定了基础。

Abstract:

This study developed ferritin-based nanoparticles carrying the African swine fever virus (ASFV) p30 protein and evaluated their immunogenicity, aiming to provide an experimental basis for the research on nanoparticle vaccines against ASFV. Initially, the gene sequences encoding the p30 protein and SpyTag were fused and inserted into the pCold-I vector to create the pCold-p30 plasmid. The gene sequences encoding SpyCatcher and ferritin were fused and then inserted into the pET-28a(+) vector to produce the pET-F-np plasmid. Both plasmids were expressed in Escherichia coli upon induction. Subsequently, the affinity chromatography-purified p30 protein was conjugated with ferritin in vitro, and the p30-ferritin (F-p30) nanoparticles were purified by size-exclusion chromatography. The morphology and structural integrity of F-p30 nanoparticles were examined by a particle size analyzer and transmission electron microscopy. Mice were immunized with F-p30 nanoparticles, and the humoral and cellular immune responses were assessed. The results showed that F-p30 nanoparticles were successfully prepared, with the particle size of approximately 20 nm. F-p30 nanoparticles were efficiently internalized by bone marrow-derived dendritic cells (BMDCs) cells in vitro. Compared with the p30 protein alone, F-p30 nanoparticles induced elevated levels of specific antibodies and cytokines in mice and stimulated the proliferation of follicular helper T cell (T_FH) and germinal center B cell (GCB) in lymph nodes as well as CD4⁺ and CD8⁺ T cells in the spleen. In conclusion, we successfully prepared F-p30 nanoparticles which significantly enhanced the immunogenicity of p30 protein, giving insights into the development of vaccines against ASFV.