融合铁蛋白的猪瘟病毒囊膜蛋白E2的表达及鉴定

目前,传统的灭活猪瘟疫苗及弱毒(减毒)猪瘟疫苗存在免疫保护期短、毒力返强等缺点,而蛋白源性亚单位疫苗可以很好的解决上述问题。铁蛋白24个亚基可以自组装成稳定的多聚体纳米颗粒,成为理想的抗原呈递载体和疫苗开发平台。基于此,将铁蛋白与具有较强免疫原性的猪瘟病毒囊膜蛋白E2(具有高保守性的保护性抗原)结合,构建融合基因E2-Fe,将该基因克隆到pET28a原核表达载体中,转化至大肠杆菌(Escherichia coli)BL21(DE3)感受态细胞中进行诱导条件的探索及其高效表达,再将表达的融合蛋白进行镍柱纯化、质谱分析、Western-blotting验证及电镜检测。结果显示,融合基因E2-Fe已成功克隆到pET-28a载体上,0.50%乳糖诱导6 h为融合蛋白E2-Fe表达的最优条件;质谱分析与Western-blotting均显示融合蛋白的大小约为51 kD,与理论值相符;电镜观察到的纳米颗粒直径约为20 nm。结果表明,融合铁蛋白的猪瘟病毒囊膜蛋白E2在大肠杆菌中得到高效表达,经镍柱纯化后可获得数量可观的自组装纳米颗粒抗原,为研究新的猪瘟疫苗拓宽了研究思路。

Expression and Identification of Swine Fever Virus Envelope Protein E2 Fusion with Ferritin

At present, the traditional inactivated and attenuated classical swine fever vaccines have some shortcomings, such as short immune protection period and strong virulence, and protein-derived subunit vaccines can solve these problems well. 24 subunits of ferritin can be self-assembled into stable multimeric nanoparticles, and become an ideal antigen presentation carrier and vaccine development platform. Based on this, ferritin was combined with classical swine fever virus envelope protein E2 (protective antigen with high conservation) to construct a fusion gene E2-Fe, which was cloned into pET28a prokaryotic expression vector and then transformed into competent cells of Escherichia coli BL21 (DE3) to explore the induction conditions and express it efficiently. Then the expressed fusion protein was purified by nickel column, analyzed by mass spectrometry, verified by Western-blotting, and detected by electron microscope. The results showed that the fusion gene E2-Fe had been successfully cloned into pET-28a vector, and the best expression condition of fusion protein E2-Fe was induced by 0.50% lactose for 6 h. Mass spectrometry analysis and Western-blotting showed that the size of the fusion protein was about 51 kD, which was consistent with the theoretical value. The diameter of nanoparticles observed by electron microscope was about 20 nm. The results showed that the envelope protein E2 of classical swine fever virus fused with ferritin was highly expressed in E. coli, and a considerable amount of self-assembled nanoparticle antigens could be obtained after purification by nickel column, which broadened the research ideas for studying new classical swine fever vaccines.