牛病毒性腹泻病毒多表位基因疫苗的构建及其免疫效果评价

Construction of the bovine viral diarrhea virus poly-epitope gene vaccine and evaluation of its immune efficacy

旨在构建牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)多表位基因与BVDV结构蛋白E0、E2基因的表达载体并检测其免疫效果。从NCBI上获得BVDV的4种具有免疫原性的结构蛋白C、E0、E2、NS3基因序列,利用生物学方法预测其B、T细胞表位;将获得的序列进行连接,重组获得新肽段(命名为AKK),通过生物学在线软件分析AKK序列的二级结构、亲水性、抗原性、三级结构;PCR分别扩增上述基因序列,构建重组表达载体GV658-AKK-E0-E2、GV658-AKK、GV658-E0、GV658-E2,并将上述构建成功的无内毒素重组质粒转染至MDBK细胞,通过蛋白免疫印迹。经PCR扩增获得2910、1170、951和729 bp大小的目的条带,与预计相符;经蛋白免疫印迹验证,在34和68 kDa处有AKK蛋白及E0-E2蛋白的融合表达;经流式细胞术验证显示,在CD3^(+)、CD4^(+)细胞占比上,共表达组极显著高于PBS组,显著高于E0组,与ELISA检测IgG抗体水平结果一致。研究表明,试验成功设计了BVDV多表位序列AKK,并成功构建真核表达载体,AKK、E0、E2蛋白高效表达,共表达组能更好地刺激机体的体液免疫和细胞免疫应答。

The aim of this study was to construct an expression vector co-expressing the poly-epitope protein and the E0E2 protein of the bovine viral diarrhea virus(BVDV),which would verify the protein expression by in vitro cell experiments.The sequences of the four immunogenic proteins,C,E0,E2,and NS3 of BVDV were obtained from NCBI,and the biological technology was used to predict their B and T cell epitopes.The sequences obtained were connected and recombined to obtain a new peptide,named AKK.The secondary structure,hydrophilicity,antigenicity,and tertiary structure of the AKK sequence were analyzed using the online biological software.PCR was used to amplify the above sequences to reconstruct recombinant expression vectors GV658-AKK-E0-E2,GV658-AKK,GV658-E0 and GV658-E2,and the above successfully constructed endotoxin-free recombinant plasmids were transfected into MDBK cells using Western blot.PCR amplification was applied and targeted bands of 2910 bp,1170 bp,951 bp and 729 bp were obtained,which were in line with the expectations.Western blot analysis confirmed that,at the sites of 34 kDa and 68 kDa,the AKK,E0,E2 proteins were co-expressed.Validation by flow cytometry showed that the co-expression group was significantly higher in CD8+than the PBS group at the CD3^(+),CD4^(+)and the E0.This was consistent with the results of the IgG antibody levels measured by ELISA.In conclusion,the BVDV poly-epitope sequence AKK was successfully designed,and the eukaryotic expression vector was successfully constructed.The AKK,E0 and E2 proteins were efficiently expressed,and the co-expression group could better stimulate the humoral and cellular immunity.