南方医科大学学报2011，Vol.31Issue(6)：955-959,5.
用于时间分辨荧光免疫分析的结核分枝杆菌培养滤液蛋白10参照品的制备
Preparation and characterization of reference samples of Mycobacterium tuberculosis culture filtrate protein-1O for time-resolved fluoroimmunoassay
摘要
Abstract
Objective To prepare reference samples of Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) and CFPlO-streptavidin fusion proteins (CFP10/SA) for time-resolved fluoroimmunoassay (TRFIA). Methods The CFP10 gene was amplified by PCR from Mycobacterium tuberculosis strain H37Rv and cloned into pET24b, pET24b-streptavidin (SA) or pET21a-SA expression vectors. The recombinant proteins CFP10, CFP10-SA and SA-CFP10 were expressed in Rosetta cells, purified via nickel affinity chromatography and refolded by dialysis. The sensitivity and stability of the resultant proteins as reference samples were evaluated by double-antibody sandwich TRFIA. Results CFP10-SA and SA-CFP10 fusion proteins were expressed as inclusion bodies, whereas CFP10 was expressed in a soluble form. The resultant purity of the 3 recombinant proteins all exceeded 95%. TRFIA results showed that CFP-SA fusion protein possessed the best sensitivity (0.02 ug/L) and stability. Conclusion The reference samples of CFP10 for TRFIA detection have been successfully prepared and can be used in the development of a diagnostic kit for Mycobacterium tuberculosis.关键词
结核分枝杆菌/培养滤液蛋白10/时间分辨荧光分析法Key words
mycobacterium tuberculosis/ culture filtrate protein 10/ time-resolved fluoroimmunoassay分类
医药卫生引用本文复制引用
郭芳芳,邹立林,吴英松,胡志明,李金龙,吕建新,高基民..用于时间分辨荧光免疫分析的结核分枝杆菌培养滤液蛋白10参照品的制备[J].南方医科大学学报,2011,31(6):955-959,5.基金项目
国家科技重大专项(2008ZX10301) （2008ZX10301）
