Cloning and expression of pab gene of M. tuberculosis isolated from pulmonary TB patient in E.coli DH5α

  • Tri Y. M. Raras
  • Diana Lyrawati
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Abstract

Background: Mycobacterium tuberculosis antigen38 is a potent serodiagnostic agent containing two M. tuberculosisspecific B-cell epitopes. The high price of imported diagnostic agents hinders realization of fast clinical TB diagnosis in developing countries. Therefore, we produced recombinant antigen38 (recAg38M) from M. tuberculosis local strain, which might be used to produce economical tuberculosis serodiagnostic kit.

Methods: Pab gene that was isolated from pulmonary TB patient in Malang was cloned into a plasmid vector (pGEMTeasy) to construct pMB38. The E.coli DH5α clone carrying pMb38 was selected on X-gal medium. The expression of pab was mediated using pPRoExHTc under the control of Trc promoter and E.coli DH5α as host.

Results: Alignment of the pab sequence from the white E.coli DH5α clones with that of M. tuberculosis H37Rv showed 98% homology. The recombinant protein in which the signal peptide has been deleted to prevent the protein being secreted into medium was found in the cytoplasm.

Conclusion: pab gene of M. tuberculosis isolated from a TB patient could be expressed in heterologous system in E.coliDH5α. (Med J Indones 2011; 20:247-54)

Keywords: Mycobacterium tuberculosis, Pab gene expression, recombinant antigen38

Author Biography

Diana Lyrawati
Department of Biochemistry-Molecular Biology, Universitas Brawijaya, Malang, Indonesia

References

  1. Chang Z, Choudary A, Lathigra R, Quiocho FA. The immunodominant 38-kDa lipoprotein antigen of Mycobacterium

  2. tuberculosis is a Phosphate-binding protein. J Biol Chem. 1994;269;1956-68.

  3. Wilkilson RJ, Haslov K, Rappuoli R, Giovannoni F, Narayanan PR, Desai CR, et al. Evaluation of the recombinant 38-kilodalton antigen of Mycobacterium tuberculosis as a potential immunodiagnostic reagent. J Clin Microbiol. 1997;35:553-7.

  4. Cheng VCC, Yew WW, Yuen KY. Molecular diagnostics in

  5. tuberculosis. Eur J Clin Microbiol Infect Dis. 2005; 24:711-20.

  6. Van der Zanden AGM. Spoligotyping, a tool in epidemiology,

  7. diagnosis and control of tuberculosis. Wageningen: Ponsen & Looijen BV; 2002.

  8. Singh M, Andersen AB, McCarthy JEG, Rohde M, Schutte

  9. H, Sanders E, et al. The Mycobacterium tuberculosis 38-kD antigen: overproduction in Escherichia coli, purification and characterization. Gene. 1992;117:53-60.

  10. Sambrook J, Fritsch E F,Maniatis T. Molecular cloning: a laboratory manual. New York: Cold Spring Harbour; 2001.

  11. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidmann JG, Sminth JA, et al. Current protocol in molecular biology. New York : Wiley and Sons; 1997.

  12. Young DB, Garbe TR. Heat shock protein and antigen of

  13. Mycobacterium tuberculosis. J Infec Immun. 2001;59: 3086-93.

  14. Yasukawa C, Kanei-Ishii T, Maekawa J, Fujimoto T, Yamamoto, Ishii S. Increase of solubility of foreign protein in E. coli by overproduction of bacterial thioredoxin. J Biol Chem. 1995;270:25328-31.

  15. Gubernator B, Seidler A, Rögner M, Szczepaniak A. Overexpression and reconstitution of a Rieske iron-sulfur protein

  16. from the higher plant. Protein Expr Purif. 2003;29:8-14.

  17. Mardining Raras TY. Study on the cyclodextrine metabolism

  18. in Klebsiella oxytoca M5a1. A molecular Approach. Saarbrucken: Dr Mueller Publisher; 2010.

Published
2011-11-01
How to Cite
1.
M. Raras TY, Lyrawati D. Cloning and expression of pab gene of M. tuberculosis isolated from pulmonary TB patient in E.coli DH5α. Med J Indones [Internet]. 2011Nov.1 [cited 2019Nov.12];20(4):247-54. Available from: http://mji.ui.ac.id/journal/index.php/mji/article/view/458
Section
Basic Medical Research