结核分枝杆菌RD1区重组11 kD蛋白的克隆表达及效力

目的在大肠杆菌中表达结核分枝杆菌RD1区11kD蛋白,并检测表达产物的效力。方法构建11kD蛋白编码基因原核表达载体,转化大肠杆菌BL21(DE3)IPTG诱导表达,离子层析柱分离纯化重组蛋白。以结核分枝杆菌致敏豚鼠进行皮肤变态反应(DTH)测定。结果结核分枝杆菌11kD蛋白在大肠杆菌中以可溶形式表达,表达蛋白占总菌体蛋白的33%以上,纯化后纯度达95%以上,并可诱导结核分枝杆菌致敏豚鼠产生迟发型超敏反应,5μg/ml11kD蛋白的效力与50IU/ml TB-PPD相当。结论重组11kD蛋白已在大肠杆菌中成功表达,并有望成为结核病皮试鉴别诊断用新试剂。

Gene Cloning, Expression and Potency of Recombinant 11 kD Protein at RD1 Region of Mycobacterium tuberculosis

Objective To express the 11 kD protein at RD1 region of Mycobacterium tuberculosis in E.coli and test for the potency of expressed product.Methods Construct the prokaryotic expression vector for 11 kD protein of Mycobacterium tuberculosis,then transform to E.coli BL21(DE3) for expression under induction of IPTG. Purify the expressed product by ion exchange chromatography and determine its potency by delayed-type hypersensitivity(DTH) test in guinea pigs sensitized with Mycobacterium tuberculosis.Results The 11 kD protein of Mycobacterium tuberculosis was expressed in a soluble form in E.coli.The expressed product contained more than 33% of total somatic protein and reached a purity of more than 95% after purification.DTH test proved that the potency of 5 μg/ml of 11 kD protein was equivalent to 50 IU/ml of TB-PPD.Conclusion Recombinant 11 kD protein at RD1 region of Mycobacterium tuberculosis was successfully expressed in E.coli and might be used as a novel reagent for the differential diagnosis of tuberculosis.