Cloning and expression of pab gene of M. tuberculosis isolated from pulmonary TB patient in E.coli DH5Î±
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 DH5Î± 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 DH5Î± as host.
Results: Alignment of the pab sequence from the white E.coli DH5Î± 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.coliDH5Î±. (Med J Indones 2011; 20:247-54)
Keywords: Mycobacterium tuberculosis, Pab gene expression, recombinant antigen38
Chang Z, Choudary A, Lathigra R, Quiocho FA. The immunodominant 38-kDa lipoprotein antigen of Mycobacterium
tuberculosis is a Phosphate-binding protein. J Biol Chem. 1994;269;1956-68.
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.
Cheng VCC, Yew WW, Yuen KY. Molecular diagnostics in
tuberculosis. Eur J Clin Microbiol Infect Dis. 2005; 24:711-20.
Van der Zanden AGM. Spoligotyping, a tool in epidemiology,
diagnosis and control of tuberculosis. Wageningen: Ponsen & Looijen BV; 2002.
Singh M, Andersen AB, McCarthy JEG, Rohde M, Schutte
H, Sanders E, et al. The Mycobacterium tuberculosis 38-kD antigen: overproduction in Escherichia coli, purification and characterization. Gene. 1992;117:53-60.
Sambrook J, Fritsch E F,Maniatis T. Molecular cloning: a laboratory manual. New York: Cold Spring Harbour; 2001.
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.
Young DB, Garbe TR. Heat shock protein and antigen of
Mycobacterium tuberculosis. J Infec Immun. 2001;59: 3086-93.
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.
Gubernator B, Seidler A, RÃ¶gner M, Szczepaniak A. Overexpression and reconstitution of a Rieske iron-sulfur protein
from the higher plant. Protein Expr Purif. 2003;29:8-14.
Mardining Raras TY. Study on the cyclodextrine metabolism
in Klebsiella oxytoca M5a1. A molecular Approach. Saarbrucken: Dr Mueller Publisher; 2010.
Copyright (c) 2011 Tri Y. M. Raras, Diana Lyrawati
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Authors who publish with Medical Journal of Indonesia agree to the following terms:
- Authors retain copyright and grant Medical Journal of Indonesia right of first publication with the work simultaneously licensed under a Creative Commons Attribution-NonCommercial License that allows others to remix, adapt, build upon the work non-commercially with an acknowledgment of the work’s authorship and initial publication in Medical Journal of Indonesia.
- Authors are permitted to copy and redistribute the journal's published version of the work non-commercially (e.g., post it to an institutional repository or publish it in a book), with an acknowledgment of its initial publication in Medical Journal of Indonesia.