猪圆环病毒2型Cap蛋白及传染性法氏囊病病毒VP2蛋白的VLPs自组装表达与免疫效力评价

Self-assembled VLPs expression and immune efficacy evaluation of Porcine circovirus type 2 Cap protein and Infectious bursal disease virus VP2 protein

为了利用同一重组大肠杆菌实现不同种动物疫病来源类病毒样颗粒(virus like particles,VLPs)的高效组装表达,试验将编码猪圆环病毒2型(PCV2)Cap蛋白和传染性法氏囊病病毒(IBDV)VP2蛋白的核苷酸序列分别克隆至pET28a-Rcodon载体中,并分别转化至改造的BL21(DE3)△lpxM tig+感受态细胞中,利用纳米粒度电位仪及透射电子显微镜分析目的蛋白表达后的胞内组装情况,并用纯化后的抗原蛋白制备的疫苗免疫动物进行免疫效力评价。结果表明:PCV2 Cap蛋白和IBDV VP2蛋白的可溶性表达水平较优化前对照组均分别提高90%左右,且目的蛋白在胞内高效自组装形成了VLPs,并排列呈均一的晶格结构;含VP2抗原的亚单位疫苗可提供100%的保护效果,含PCV2抗原的亚单位疫苗的抗体水平较高,显著高于阴性对照组纯化后的抗原蛋白制备的疫苗(P<0.05)。说明PCV2 Cap蛋白和IBDV VP2蛋白在BL21(DE3)△lpxM tig+感受态细胞中均可实现高效可溶性表达与细胞内VLPs自组装,且免疫原性良好,能够给免疫动物提供较好的免疫保护。

In order to achieve efficient assembled and expression of virus like particles(VLPs)from different animal epidemics in the same recombinant Escherichia coli,in this study,the nucleotide sequences encoding Porcine circovirus type 2(PCV2)Cap protein and Infectious bursal disease virus(IBDV)VP2 protein were codon-optimized and cloned into pET28a-Rcodon vector before transformed into BL21(DE3)△lpxM tig+host strain,respectively.The intracellular assemblies of the target proteins were analyzed by nano particle size analyzer and transmission electron microscope,and the vaccines prepared by the purified antigens were used to evaluate the efficacy of immunizing animals.The results showed that the soluble expression level of PCV2 Cap and IBDV VP2 proteins were increased about 90%compared with the control group before optimization respectively,and the target proteins were efficiently self-assembled into VLPs intracellularly and arranged in a uniform lattice structure.The subunit vaccine containing VP2 antigen could provide 100%protective effect.The antibody level of subanit vaccine containing PCV2 antigen was higher than that of negative control group.These results indicated that both PCV2 Cap protein and IBDV VP2 protein were highly soluble expressed and self-assembled into VLPs intracellularly in recombinant Escherichia coli with good immunogenicity,and it could provide better immune protection for immunized animals.