嗜水气单胞菌外膜蛋白ompTS基因的高效表达及其免疫原性

The expression of Aeromonas hydrophila ompTS gene and the immunogenicity of recombinant OmpTS

根据嗜水气单胞菌外膜蛋白基因ompoTS的核苷酸序列设计引物,运用聚合酶链式反应(PCR)扩增出与预期大小相符的基因片段。将此基因片段克隆至质粒pRSET A的BamHI和EcoRI位点,构建重组质粒,转化大肠杆菌BL21(DE3),经IPTG诱导获得高效表达,SDS-PAGE蛋白电泳表明在39.9kD处出现超强特异带,占总蛋白的51%。以Ni-NTA-Conjugate抗体进行Western blot分析证明该39.9kD的蛋白为所表达的融合蛋白。纯化融合蛋白注射雄性新西兰大白兔可诱导产生特异抗体。ELISA和Western blot检测结果显示,该抗体与表达的融合蛋白和嗜水气单胞菌中提取的36.9kD外膜蛋白均呈阳性反应,表明所表达的融合蛋白仍保持原有外膜蛋白的免疫原性,从而为此融合蛋白作为疫苗的候选成份提供理论依据。

Primers were designed based on ompTS gene reported recently. With the specific primers, one target fragment about 1024bp lacking the signal sequence of ompTS gene was amplified from A. hydrophila genomic DNA via PCR. The ompTS gene was hyperexpressed using gene fusion expression vector pRSET system, and the recombinant OMP exhibited a size cf 39.9kD with SDS-PAGE and Wastem blot analysis, which showed about 51% of total lysate proteins. Antibody to the pure recombinant OMP reacted not only with the pure recombinant OMP but also with the purified OMPs from A. hydrophila in ELISA and the 36.9kD OMP in Western blot. The result indicates that recombinant OMP has the same epitope with the nature one.