Tuberc Respir Dis.  2006 Sep;61(3):239-247. 10.4046/trd.2006.61.3.239.

The Purification and Immunogenicity of TB-14 Recombinant Protein of Mycobacterium tuberculosis

• Song HY
• Kim YH
• Kim CH
• Min YK
• Kim DJ
• Ko KK

• KMID: 1970263
• DOI: http://doi.org/10.4046/trd.2006.61.3.239

Abstract

BACKGROUND: Culture filtrate proteins secreted by mycobacteria are thought to play an important role in inducing protective immunity and to develop new methods for diagnosing tuberculosis.
METHODS
A culture filtrate protein of M. avium that was strongly reactive with goat antiserum against M. intracellulare was constructed. Its homologous protein (TB-14) in M. tuberculosis was cloned, expressed and purified. The inductions of IFN-gamma stimulated with 10 microgram of TB-14 recombinant protein and 10 microgram PPD were estimated by using whole bloods from seven PPD (-) subjects, seven PPD (+) healthy volunteers and nine tuberculosis patients.
RESULTS
M. avium culture filtrate protein was confirmed as a hypothetical protein that was termed contig 116. A novel 14-kDa recombinant protein (TB-14) of M. tuberculosis was composed of 148 amino acids, including 30 amino acids of the signal peptide, and it showed 78% homology with M. avium. In the PPD (+) healthy volunteers, recombinant TB-14 protein strongly induced the secretion of IFN-gamma in whole blood cultures.
CONCLUSION
These results suggest that TB-14 recombinant protein might play an important role in inducing cell-mediated immunity against tuberculosis. Furthermore, TB-14 protein antigen and its antiserum will be available for the development of new diagnostic tools for tuberculosis.

Keyword

Tuberculosis; TB-14 recombinant protein; IFN-gamma

MeSH Terms

Amino Acids
Clone Cells
Goats
Healthy Volunteers
Humans
Immunity, Cellular
Mycobacterium tuberculosis*
Mycobacterium*
Protein Sorting Signals
Tuberculosis
Amino Acids
Protein Sorting Signals

Figure

• 

  Figure 1 Western blot of culture filtrates of M. tuberculosis(TB) and M. avium(AVI) using M. intracellulare antiserum. Left two lanes; antiserum before pre-absorption with M. tuberculosis culture filtrates, Right two lanes; antiserum after pre-absorption.

• 

  Figure 2 Simultaneous two color protein stain (red) and western blot (blue) following preparative IEF and 2-D gel electrophoresis of M. avium filtrate.

• 

  Figure 3 The amino acid sequences of three homologs.

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  Figure 4 Expression of 6His-tagged TB-14 protein was induced with 1 mM IPTG. Proteins were visualized by Coomassie stain. M: marker, 1; flow-through, 3: wash, 3-8: TB-14 elutes(left). After Prep Cell purification, only single protein bands are shown around 14 kDa (right).

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  Figure 5 The concentrations of IFN-γ induced by PPD and TB-14 recombinant protein in the supernatant of diluted whole bloods from PPD(-), PPD(+) healthy volunteers, and TB patients.

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