大肠杆菌 LT B亚基基因表达载体对犬细小病毒VP2 DNA疫苗免疫应答的增强作用

Escherichia coli heat-labile enterotoxin B subunit enhances the immune response against canine parvovirus VP2 in mice immunized by VP2 DNA vaccine

摘要:

摘要：【目的】 通过融合基因表达载体和共免疫基因表达载体研究大肠杆菌不耐热肠毒素（LT）B亚基基因对犬细小病毒VP2 DNA疫苗免疫应答的影响。【方法】提取大肠杆菌44815菌株基因组DNA，通过PCR方法从基因组DNA中扩增LTB基因，同时采用PCR方法从含有犬细小病毒VP2基因的质粒中扩增VP2的主要抗原表位基因（VP2-70，编码70个氨基酸）。将上述基因分别连接到含有人CD5信号肽序列的载体pcDNA-CD5sp上，分别构建成它们的分泌型真核表达载体，pcDNA-CD5sp-LTB 和pcDNA-

Abstract:

Abstract: [Objective] To investigate the effect of Escherichia coli heat-labile enterotoxin (LT) B subunit (LTB) gene on canine parvovirus (CPV) VP2 gene vaccine. [Methods] The LTB gene was amplified by PCR from genomic DNA of E. coli 44815 strain. The VP2-70 fragment (210 bp) encoding major epitope of VP2 (70 amino acids) was amplified by PCR from a plasmid encoding VP2 gene. VP2-70 and LTB genes were inserted into the eukaryotic vector to construct VP2-70 gene, LTB gene and VP2-70-LTB fused gene vectors. The mice were immunized with VP2-70 vector, VP2-70-LTB fused vector, or VP2-70 vector plus LTB vector, respectively. The antibody titers at the different time were measured by using ELISA method. The spleen lymphocyte proliferation activity was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide（MTT）assay. [ Results ] The sequence of VP2-70 and LTB genes was identified. The recombinant VP2-70 and LTB proteins could be expressed in HEK293T cells in a secretory manner. The mice immunized with VP2-70 vector, VP2-70-LTB vector or VP2-70 vector plus LTB vector could generate the specific antibody against VP2 protein. The antibody titer immunized with VP2-70-LTB vector reached 1:5120 at 35 d post immunization, significantly higher than that of other two groups (P<0.01). For antibody isotype analysis, the IgG1 isotype antibody titers in all test groups were significantly higher than of IgG2a (P<0.01). The high-level spleen lymphocyte stimulation index was observed in the three test groups under the stimulation with Con A, higher than that in control groups (P<0.01). [ Conclusion] LTB gene could enhance the humoral immune response of CPV VP2 gene vaccine in mice.