摘要
根据嗜水气单胞菌外膜蛋白基因ompTS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pRSETA的BamHI和EcoRI位点 ,构建重组质粒 ,转化大肠杆菌BL2 1(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5 1%。以Ni NTA Conjugate抗体进行Westernblot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Westernblot检测结果显示 ,该抗体与表达的融合蛋白和从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 。
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 of 39.9kD with SDS-PAGE and Western blot analysis, which showed about 51% of total lysate proteins. Antibody to the purified recombinant OMP reacted not only to the recombinant OMP but also to the purified OMPs from A. hydrophila in ELISA and the 36.9kD OMP in Western blot. The result indicates that the recombinant OMP has the same epitope with the nature one.
出处
《生物工程学报》
CAS
CSCD
北大核心
2002年第3期300-303,共4页
Chinese Journal of Biotechnology
基金
国家自然科学基金资助 (No 30 0 0 0 12 8)~~
