口蹄疫病毒三价复合多表位佐剂DNA疫苗构建及其免疫原性

Molecular design and immunogenicity of a multiple-epitope foot-and-mouth disease virus antigen, adjuvants, and DNA vaccination

摘要:

以O型、A型口蹄疫病毒(FMDV)结构蛋白VP1全基因和AsiaI型FMDV两个基因拓扑型的结构蛋白VP1基因上的5个抗原表位基因作为主要免疫原基因, 以来源于非结构蛋白3ABC和结构蛋白VP4上的3个Th2细胞表位基因作为辅助基因, 构建了O型、A型和Asia1型FMDV复合多表位基因工程疫苗表达盒OAAT, 在此基础上, 以金黄色葡萄球菌肠毒素A(SEA)为基因佐剂, 通过分子设计构建了SEA与OAAT融合表达基因。将构建好的表达盒OAAT与SEA融合表达基因克隆至真核表达载体PVAX1 PCMV启动子下游, 构建了口蹄疫三价基因佐剂DNA疫苗pEA。经Western blotting和IFA 检测, 目的蛋白在Hela细胞中获得正确表达。小鼠免疫实验表明, pA和pEA免疫组的血清抗体均能分别与O型、A型和AsiaI抗原反应, 与对照组相比差异较显著, 且pEA免疫组和灭活疫苗免疫组抗体水平均显著高于pA免疫组; 同时pA和pEA免疫小鼠细胞因子IL-2、IFN-g、IL-4和IL-10较对照组显著提高, 且pEA免疫组的IL-2、IFN- g和IL-4水平明显高于pA免疫组。用O/NY00和Asia1/YNBS/58株FMDV进行豚鼠攻毒免疫保护试验, 结果表明O型和Asia1型FMDV二联灭活疫苗免疫组均获得完全保护, pA免疫组均获得2/4保护, pEA免疫组均获得3/4保护, 而pVAX1和PBS免疫组完全没有保护。实验结果表明, SEA与OAAT融合表达蛋白具有较好免疫原性, 且SEA可以作为DNA疫苗的有效基因佐剂。

Abstract:

We designed and constructed a fuse expression gene OAAT and SEA (staphylococcal enterotoxin A) on the basis of the OAAT designed and constructed which consists of the structural protein VP1 genes from serotypes A and O FMDV, 5 major VP1 immunodominant epitopes from two genotypes of Asia1 serotype, and 3 Th2 epitopes originating from the non-structural protein, 3ABC gene and structural protein VP4 gene. The recombinant plasmids pEA was constructed using Staphylococcal enterotoxin A (SEA) as a genetic adjuvant. Expressions of target gene from the pEA in Hela cell were verified by IFA and Western blotting. The experiment of BALB/c mice immunized with the DNA vaccines showed that pA and pEA could induce simultaneously specific antibodies against serotypes A, Asia1, and O FMDV, and the highest antibody titres were found in the pEA and inactivated vaccine groups compared to pA vaccinating mice. Compared with the control, the levels of IL-2, IFN-γ, IL-4, and IL-10 expression by splenic lymphocytes from mice immunized with pA and pEA were significantly increased. In addition, we found that the levels of IL-2, IFN-γand IL-4 from the mice immunized with pEA was higher than mice immunized with pA did. The results of viral challenge in guinea pigs showed the pA, pEA and inactivated vaccine provided full protection in 2/4, 2/4, 3/4, 3/4 and 4/4, 4/4 guinea pigs from challenge with FMDV O/NY00 and Asia1/YNBS/58, respectively. The results demonstrated fuse protein OAAT and SEA may be potential immunoge against FMDV, furthermore, SEA may be an effective genetic adjuvant for DNA vaccine.