Preparation and characterization of reference samples of Mycobacterium tuberculosis culture filtrate protein-10 for time-resolved fluoroimmunoassay / 南方医科大学学报
Journal of Southern Medical University
;
(12): 955-959, 2011.
Article
in Chinese
| WPRIM
| ID: wpr-332508
ABSTRACT
<p><b>OBJECTIVE</b>To prepare reference samples of Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) and CFP10-streptavidin fusion proteins (CFP10/SA) for time-resolved fluoroimmunoassay (TRFIA).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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 µg/L) and stability.</p><p><b>CONCLUSION</b>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.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Reference Standards
/
Bacterial Proteins
/
Fluoroimmunoassay
/
Gene Amplification
/
Genetics
/
Methods
/
Mycobacterium tuberculosis
Language:
Chinese
Journal:
Journal of Southern Medical University
Year:
2011
Type:
Article
Similar
MEDLINE
...
LILACS
LIS