结核分枝杆菌重组蛋白的制备及抗原性分析

Preparation and antigenicity assay of recombinant proteins of Mycobacterium tuberculosis

目的:克隆表达结核分枝杆菌Mr16×103和Mr38×103重组蛋白,测定其抗原性和特异性.方法:利用聚合酶链反应自结核分枝杆菌基因组DNA扩增Mr16×103和Mr38×103抗原基因,经测序鉴定后克隆于pGEX4T2表达载体,转化于大肠杆菌BL21菌株进行诱导表达,利用GSTrapFF蛋白柱纯化表达产物,通过酶联免疫吸附测定(ELISA)检测其抗原性与特异性.结果:携带重组质粒的菌株经诱导产生的表达量分别为42%和18%;Mr16×103和Mr38×103蛋白量分别为:688mg/L和217mg/L.其中Mr16×103蛋白作为包被抗原ELISA法检测的灵敏度为67.0%,特异性为100%,准确性为84.0%;Mr38×103蛋白作为包被抗原ELISA检测的灵敏度为79.8%,特异性为99%,准确性为89.7%.结论:以Mr16×103,Mr38×103重组蛋白为抗原具有良好的抗原性和特异性,可用于结核分枝杆菌诊断方法的建立及临床诊断.

AIM： To clone and express the Mr 16 ×10^3 and Mr 38 ×10^3 proteins of Mycobacterium tuberculosis in E. coli, and to characterize its antigenicity and specificity. METHODS： The Mr 16 ×10^3 and Mr 38 ×10^3 antigen genes were amplified from Mycobacterium tuberculosis genome DNA by polymerase chain reactions and cloned into pGEX-4T-2 expression vector after sequencing. BL21 strain of E. coli was transformed with the recombinant vectors and induced to express recombinant proteins. The proteins were purified by affinity column chromatography. The antigenicity and specificity of purified proteins were estimated by enzymelinked immunoabsorbant assay. RESULTS： The BL21 strains of E. coli with recombinant vectors showed 42% and 18% of Mr 16 ×10^3 （688 mg/L） and Mr 38 ×10^3（217 mg/L） gene expressions after induction. The sensitivity of Mr 16 ×10^3 and Mr 38 ×10^3 proteins were 67.0% and 79. 8%, specificity were 100% and 99% ,accuracy were 84.0% and 89. 7% respectively. CONCLUSION： The expressions and purifications of recombinant Mr 16 ×10^3 and Mr 38 ×10^3 antigens with good antigenicity and specificity facilitate their research and clinical application in the diagnosis of Mycobacterium tuberculosis.