| 摘要: |
| 【目的】利用大肠杆菌原核表达系统表达猪圆环病毒2型Cap蛋白及在其C-端嵌合PRRSV T细胞抗原表位的Cap嵌合蛋白，对Cap蛋白形成的病毒样颗粒（VLPs）的稳定性、免疫原性以及C-端嵌合PRRSV T细胞抗原表位VLPs的热稳定性进行研究，为猪圆环病毒2型(PCV2)VLPs疫苗的高效制备及基于Cap VLPs纳米骨架展示技术嵌合疫苗的开发奠定基础。【方法】采用分子生物学方法合成PCV2b型强毒株ZJ的cap基因，在其C-端插入PRRSV T细胞抗原表位T1、T2、T3、T4、T5,构建cap-T1～cap-T5基因；将cap及cap-T1～cap-T5基因与pET28a表达载体连接，构建重组表达载体，转染大肠杆菌ClearColiTMBL21(DE3)进行诱导表达，并对表达产物的热稳定性及免疫效果进行检测。【结果】实现了Cap蛋白及其C-端嵌合PRRSV T细胞抗原表位的Cap-T1、Cap-T2、Cap-T3、Cap-T4和Cap-T5嵌合蛋白在大肠杆菌中的可溶性表达，并成功观察到了VLPs；表达产物热稳定性分析显示，在60 ℃处理30 min条件下，Cap VLPs保持稳定，而C-端嵌合PRRSV T细胞抗原表位的VLPs热稳定性大幅下降；小鼠免疫效果检测显示，Cap VLPs诱导产生了高水平特异性抗体，其中VLPs+佐剂S350组效果最好。【结论】利用大肠杆菌表达系统表达的PCV2 Cap蛋白可高效自组装成耐热和高免疫原性的VLPs，但在Cap蛋白C-端嵌合PRRSV T细胞抗原表位后的嵌合VLPs组装效果和稳定性急剧下降，提示PCV2 VLPs作为纳米骨架用于传染病疫苗研发仍有较大局限性。 |
| 关键词: 猪圆环病毒；Cap蛋白；C-端嵌合；病毒样颗粒；热稳定性 免疫原性 |
| DOI： |
| 分类号: |
| 基金项目:“十三五”涉农专项（2016YFD0500306）；国家自然科学基金项目（31572556） |
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| Effect of C-terminal chimeric PRRSV T cell epitopes on stability of PCV2 VLPs |
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LIU Xiangyu,NIE Qingqing,DU Enqi,et al
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| Abstract: |
| 【Objective】To lay foundation for the efficient preparation of PCV2 VLPs vaccine and the development of chimeric vaccine based on Cap VLPs nanoskeleton display technology,porcine circovirus type 2 Cap protein and its chimeric protein were expressed by Escherichia coli prokaryotic expression system and stability and immunogenicity of VLPs were studied.【Methed】The Cap protein of PCV2b ZJ (Nanjing) was expressed in ClearColiTMBL21(DE3).On this basis,PRRSV T cell epitopes of T1,T2,T3,T4,and T5 were inserted into the C-terminal of cap gene to construct cap-T1,cap-T2,cap-T3,cap-T4,and cap-T5 genes.Then,the recombinant expression vector was constructed and transfected into ClearColiTMBL21(DE3),and the thermal stability and immune effect of the expressed product were detected.【Result】The soluble expression of Cap,Cap-T1,Cap-T2,Cap-T3,Cap-T4,and Cap-T5 in ClearColiTMBL21(DE3) was obtained and VLPs were successfully observed.The thermal stability of the expressed product showed that VLPs remained stable at 60 ℃ for 30 min,while the thermal stability of chimeric VLPs decreased significantly.Immunoassay results showed that Cap VLPs induced high level of Cap specific antibodies in mice,and the vaccine group (VLPs+adjuvant S350) had the highest specific antibody level.【Conclusion】The PCV2 Cap protein expressed by ClearColiTMBL21(DE3) can be highly self-assembled into heat-resistant and immunogenicity VLPs.However,the assembly efficiency and stability of chimeric VLPs decreased sharply after the C-terminal chimeric PRRSV T cell epitope,suggesting that PCV2 VLPs has major limitations as nano skeleton in the research and development of infectious disease vaccine. |
| Key words: porcine circovirus Cap protein C-terminal chimeric VLPs thermostable immunogenicity |
