猪圆环病毒3型Cap重组鞭毛蛋白融合表达及其免疫原性研究

Fusion Expression and Immunogenicity of Recombinant Cap-flagellin Protein of Porcine Circovirus Type 3

【目的】利用重组杆状病毒系统表达猪圆环病毒3型(Porcine circovirus type 3,PCV3)Cap重组鞭毛蛋白并研究该蛋白的反应原性和免疫原性,为开发PCV3亚单位疫苗提供数据支持。【方法】通过对Cap基因进行最适密码子优化并与鼠伤寒沙门氏菌鞭毛蛋白基因Flagellin进行融合后克隆到pFastBac TMⅠ载体。通过Bac-to-Bac系统,筛选获得重组Cap-Flagellin杆状病毒,利用SDS-PAGE、间接免疫荧光试验(indirect immunofluorescence assay,IFA)和Western blotting鉴定重组杆状病毒,将重组Cap-Flagellin杆状病毒免疫小鼠评价其免疫原性。【结果】重组Cap-Flagellin杆状病毒在Sf9细胞中能有效表达重组蛋白,SDS-PAGE检测可见68 ku的目的条带。IFA结果显示,重组Cap-Flagellin杆状病毒感染后72 h在Sf9细胞膜及胞浆中均出现绿色特异性荧光;Western blotting分析表明,重组蛋白能与PCV3阳性血清发生特异性反应,表明重组Cap-Flagellin蛋白具有良好的反应原性。小鼠免疫试验结果表明,与Cap杆状病毒组相比,重组Cap-Flagellin杆状病毒免疫小鼠2l和28 d后可诱导产生更高水平的Cap特异性抗体(P<0.05;P<0.01),且Cap-Flagellin杆状病毒组小鼠脾细胞白细胞介素-8(IL-8)细胞因子水平显著或极显著高于其他组(P<0.05;P<0.01),表明重组Cap-Flagellin蛋白具有良好的免疫原性。【结论】本研究利用杆状病毒表达系统成功表达了PCV3 Cap重组鞭毛蛋白,并证实其具有良好的生物学活性,为PCV3亚单位疫苗的开发研制奠定了基础。

【Objective】The aim of this experiment was to express the recombinant Cap-Flagellin protein of Porcine circovirus type 3(PCV3)by Baculovirus expression system and analyze its reactogenicity and immunological properties,providing important support for the development of PCV3 subunit vaccine.【Method】The Cap gene was optimized by optimal codon and fused with Flagellin gene of Salmonella Typhimurium then cloned into pFastBac TMⅠvector.Recombinant Cap-Flagellin Baculovirus was rescued through the Bac-to-Bac system and identified by SDS-PAGE,indirect immunofluorescence assay(IFA)and Western blotting,and then mice were immunized with recombinant Cap-Flagellin Baculovirus to evaluate its immunogenicity.【Result】Recombinant Cap-Flagellin Baculovirus could effectively express recombinant proteins in Sf9 cells,the target protein bands of 68 ku were detected by SDS-PAGE.IFA results showed that the specific green fluorescence occurred in the membrane and cytoplasm of Sf9 cells 72 h after recombinant Baculovirus infection.Western blotting showed that the recombinant protein could react specifically with PCV3 positive serum,all indicated it had good reactogenicity.The serum Cap-specific antibody levels of mice immunized with Cap-Flagellin Baculovirus on the 21th and 28th day were higher compared with the Cap Baculovirus group(P<0.05 or P<0.01),and IL-8 cytokine level of spleen cells in recombinant Cap-Flagellin group was significantly or extremely significantly higher than that of other groups(P<0.05 or P<0.01),which indicated that the recombinant Cap-Flagellin protein had better immunogenicity.【Conclusion】In this study,PCV3 Cap recombinant flagellin was successfully expressed by Baculovirus expression system and had good biological activity which laid a good foundation for the development of PCV3 subunit vaccine.