Preparation and characterization of reference samples of Mycobacterium tuberculosis culture filtrate protein-10 for time-resolved fluoroimmunoassay.
- Author:
Fang-fang GUO
1
,
2
;
Li-lin ZOU
;
Ying-song WU
;
Zhi-ming HU
;
Jin-long LI
;
Jian-xin LV
;
Ji-min GAO
Author Information
- Publication Type:Journal Article
- MeSH: Bacterial Proteins; genetics; standards; Fluoroimmunoassay; methods; Gene Amplification; Mycobacterium tuberculosis; isolation & purification; Reference Standards
- From: Journal of Southern Medical University 2011;31(6):955-959
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo prepare reference samples of Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) and CFP10-streptavidin fusion proteins (CFP10/SA) for time-resolved fluoroimmunoassay (TRFIA).
METHODSThe 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.
RESULTSCFP10-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 µg/L) and stability.
CONCLUSIONThe 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.
