【摘要】 以牛分枝杆菌ValleeⅢ株基因组DNA为模板扩增hsp65和esat6基因,采用重叠延伸剪接技术(SOE)获得了融合基因hsp65-esat6,将hsp65-esat6连接到原核表达载体pET32a(+)上,构建重组质粒pET65-E6,将其转化到大肠杆菌BL21(DE3)感受态细胞中,以IPTG诱导(终浓度为1 mmol/L),表达产物进行SDS-PAGE分析。以Ni2+亲和层析柱纯化表达的融合蛋白和Western-blot分析该融合蛋白的免疫活性。结果表明:Hsp65-ESAT6融合蛋白以包涵体形式表达。表达的融合蛋白裂解为两部分,即58 ku和30ku,二者相加与预测大小88 ku相符。纯化的融合蛋白能与抗牛分枝杆菌阳性血清发生反应。更多还原

【Abstract】 The DNA fragments of hsp65 and esat6 were amplified separately by PCR from genomic DNA of Mycobacterium bovis Vallee Ⅲ strain,and a fusion gene hsp65-esat6 was obtained by SOE(splicing-by-overlapping extension).Then the fusion gene was inserted into pET32a(+) to construct a recombinant plasmid pET65-E6.The recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells under induction of 1mmol/L IPTG.SDS-PAGE analysis showed that the expressed fusion protein was insoluble.It was separated into two proportions,namely 58ku and 30ku.The total protein molecular mass of the fusion protein was in accordance with the expected 88ku.Western blot analysis showed that the protein purified by Ni2+ Purification System could be recognized by sera from bovines infected with M.bovis.更多还原