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Song, Kim, Kim, Min, Kim, and Ko: The Purification and Immunogenicity of TB-14 Recombinant Protein of Mycobacterium tuberculosis
Original Article
Tuberculosis and Respiratory Diseases

Tuberculosis and Respiratory Diseases 2006; 61(3): 239-247.

Published online: 30 September 2006

DOI: https://doi.org/10.4046/trd.2006.61.3.239

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

Ho-Yeon Song, M.D.1, Young-Hee Kim, M.D.1, Chang-Hwan Kim, M.D.1, Young-Ki Min, M.D.2, Dae-Joong Kim, M.D.3, Kwang-Kjune Ko, M.D.1

1Department of Microbiology, College of Medicine, Soonchunhyang University, Cheonan, Korea.

2Department of Physiology, College of Medicine, Soonchunhyang University, Cheonan, Korea.

3Department of Pathology, Cheongju St. Mary Hospital, Cheongju City, Chungbuk, Korea.

Address for correspondence: Ho Yeon Song, M.D., Ph.D. Department of Microbiology, College of Medicine, Soonchunhyang University, Cheonan, 330-090, Korea. Phone: 041-570-2412, Fax: 041-577-2415, songmic@sch.ac.kr

Received 19 July 2006       Accepted 28 August 2006

Copyright © 2006 The Korean Academy of Tuberculosis and Respiratory Diseases

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-γ stimulated with 10 µg of TB-14 recombinant protein and 10 µg 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-γ 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.

Keywords: Tuberculosis, TB-14 recombinant protein, IFN-γ

Figures and Tables

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