布鲁氏菌BP26蛋白的原核表达及鉴定

Prokaryotic Expression and Identification of Brucella BP26 Protein

为了实现布鲁氏菌BP26蛋白在原核宿主细胞进行高效表达,对布鲁氏菌BP26基因进行密码子优化,化学合成了BP26全基因,并将其克隆至pET30a(+)载体上,构建重组质粒pET30a(+)-bp26,转化至基因工程菌BL21(DE3),利用IPTG进行诱导表达。利用His-tag镍柱纯化BP26重组蛋白,并对纯化后的重组蛋白进行鉴定。结果显示:重组质粒经Nde I与Xhol I双酶切,酶切产物通过琼脂糖凝胶电泳显示与目的产物条带大小一致;当诱导条件为IPTG浓度1 mmol/L,37℃诱导8 h时,重组蛋白的表达量最高;纯化后的目的蛋白经过Western Blot鉴定,显示在27.8 kD左右有一条明显的蛋白印迹条带,与预期大小相符。本研究成功实现了在大肠杆菌高效表达BP26蛋白,为后续布病检测新试剂的研制及疫苗的研发奠定了生物学基础。

In order to achieve the high expression of Brucella BP26 protein in E.coli,the gene sequences of bp26 were optimized and synthesized,then theywere cloned into the pET30a(+)vector to construct recombinant plasmid pET30a(+)-bp26,which was transformed into E.coli BL21,then IPTG was used as an inducer to induce the expression of recombinant protein.The recombinant BP26 protein was purified by Ni affinity chromatography,then analyzed by SDS-PAGE and Western Blot.The results showed that when the recombinant E.coli was induced by 1mM IPTG at 37℃for 8 hours,the BP26 protein was the highest.SDS-PAGE showed that the purity of BP26 protein is over 90%,Western Blot showed an obvious band at 27.8 kD,in line with the expected.The BP26 protein was highly expressed in E.coli,which established the biological foundation for developing new reagent for detection of brucellosis and developing new vaccinesofBrucella.