O型口蹄疫病毒VP1基因与大肠杆菌不耐热肠毒素LTB 基因的融合表达及表达产物的免疫原性分析

Fusion expression of Escherichia coli heat-labile enterotoxin B subunit gene and foot-and-mouth disease virus type O VP1 gene and immunogenicity analysis

摘要:

以质粒pMDTLT为模板、用PCR的方法扩增出LTB基因, 然后将其插入到pETVP1质粒中VP1基因的上游,构建了含有融合基因LTBVP1的表达质粒pETLTBVP1。转化宿主菌BL21(DE3)LysS后进行诱导表达, 诱导菌经SDS-PAGE显示重组蛋白以包涵体的形式表达, 分子量约为39 kD; Western blotting分析表明, 重组蛋白能与FMDV阳性血清及兔抗霍乱毒素(CT)血清反应, 说明融合蛋白保持了LTB和VP1各自的免疫学活性。小鼠免疫实验表明: 该融合蛋白通过腹腔接种小鼠能诱导产生较强的免疫应答反应, 免疫鼠产生的血清抗体水平高于试验中商品口蹄疫疫苗免疫组。

Abstract:

LTB gene fragment was amplified by PCR from plasmid pMDTLT, and a recombinant plasmid pETLTBVP1 was constructed by inserting LTB gene fragment into VP1 gene expression plasmid pETVP1 constructed previously. The recombinant plasmids were transformed into E. coli BL21(DE3) and induced to express by IPTG. The recombinant protein existed in the inclusion body and its molecular weight was about 39 kD proved by SDS-PAGE analysis. Further Western blotting showed that the fusion protein could be reacted with both anti-FMDV and anti-cholera toxin serum demonstrating the immunoactivity of the fusion protein. Strong immune responses can be induced in mice inoculated with the fusion protein intraperitoneally, and the serum antibody level is higher than that of commercial foot-and-mouth disease vaccines.