Molecular Cloning, Expression and Purification of Protein TB10.4 Secreted by Mycobacterium Tuberculosis

Document Type : Original Article


1 Microbiology & Virology Research Centre, Bu- Ali Research Institute, Mashad University of Medical Sciences, Mashad, Iran

2 Division of Immunobiochemistry, Immunology Research Centre, Bu- Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran


Tuberculosis (TB) is the leading cause of mortality among the infectious diseases, especially in developing countries. One of the main goals in tuberculosis research is to identify antigens which have the ability of inducing cellular and/or humoral immunity in order to use them in diagnostic reagents or vaccine design. The aim of this study was to clone and express the TB'0.4 protein in Escherichia coli expression system. Materials and Methods
DNA was extracted from Mycobacterium tuberculosis H37Rv. Gene specific primers were designed using Gene Runner software according to sanger sequence database. Gene tb'0.4 fragment was amplified by PCR method and purified tb'0.4 gene was cloned into pET 102/D vector. Plasmid containing pET102/D-10.4 was transformed into competence E. coli TOP'0. A positive transformant was chosen and plasmids DNA was isolated and subsequently transformed into competence E. coli BL2'(DE3). The bacterium was induced by IPTG and its lysates were loaded directly onto SDS-PAGE. Purified recombinant protein was achieved using metal affinity chromatography (Ni-nitrilotriacetic acid).
TB'0.4 molecule was successfully cloned, expressed, and purified. An approximately 26.4 kDa exogenous protein was observed on the SDS-PAGE. The recombinant protein was confirmed by DNA sequencing of correct insert.
The success of expressing the TB'0.4 protein could serve as a basis for further studies on the usefulness of the gene and its expression product in the development of subunit vaccine and diagnostic method.


1.Bloom BR, Murray CJ. Tuberculosis: commentary on a emergent killer. Science 1992; 257:1055-1064.
2.Global tuberculosis control-surveillance, planning, financing. WHO report. 2006. Available at: http// Accessed Jan 2008.
3.Doherty TM. Real world TB vaccines: clinical trials in TB-endemic regions. Vaccine 2005; 23:2109-2114.
4.Wang J, Xing Z. Tuberculosis vaccines: the past, present and future. Expert Rev Vaccines 2002; 1:341-354. Review.
5.Weldingh k, Rosenkrand I, Okkles LM, Doherty TM, Andersen P. Assessing the serodiagnostic potential of 35 Mycobacterium tuberculosis proteins and identification of four navel serological antigens. J Clin Microbiol 2005; 43:57-65.
6. Young DB, Kaufmann SH, Hermans PW, Thole JE. Mycobacterial protein antigens: a compilation. Mol. Microbiol. 1992; 6:133-145.
7.Andersen P, Askgaard D, Ljungqvist L, Bentzon MW, Heron I. T-cell proliferative response to antigens secreted by Mycobacterium tuberculosis. Infect Immun 1991; 9:1558-1563.
8.Andersen P, Heron I. Specificity of a protective memory immune response against Mycobacterium tuberculosis. Infect Immun 1993; 61:844-851.
9.Horwitz MA, Lee BW, Dillon BJ, Harth G. Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis. Proc Natl Acad Sci USA 1995; 92:1530-1534.
10.Hubbard RD, Flory CM, Collins FM, Cocito C. Immunization of mice with mycobacterial culture filtrate proteins. Clin Exp Immunol 1992; 88:94-98.
11.Orme IM, Andersen P, Boom WH. T cell response to Mycobacterium tuberculosis. J Infect Dis 1993; 167:1481-1497.
12.Roberts AD, Sonnenberg MG, Ordway DJ, Furney SK, Brennan PJ, Belisle JT, et al. Characteristics of protective immunity engendered by vaccination of mice with purified culture filtrate protein antigens of Mycobacterium tuberculosis. Immunology 1995; 85:502-508.
13.Haslov K, Andersen A, Nagai S, Gottschau A, Sorensen T, Andersen P. Guinea pig cellular immune responses to proteins secreted by Mycobacterium tuberculosis. Infect Immun 1995; 63:804-810.
14.Bassey EO, Catty D, Kumararatne DS, Raykundalia C. Candidate antigens for improved serodiagnosis of tuberculosis. Tuber Lung Dis 1996; 77:136-145.
15.Havlir DV, Wallis RS, Boom WH, Daniel TM, Chervenak K, Ellner JJ. Human immune response to Mycobacterium tuberculosis antigens. Infect Immun 1991; 59:665-670.
16.Kato-Maeda M, Rhee JT, Gingeras TR, Salamon H, Drenkow J, Smittipat N, et al. Comparing genomes within the species Mycobacterium tuberculosis. Genome Res 2001;11:547-554.
17.Skjot RLV, Brock I, Arend SM, Munk ME, Theisen M, Ottenhoff THM, et al. Epitope mapping of the immunodominant antigen TB10.4 and the two homologous proteins TB10.3 and TB12.9 which constitute a subfamily of the esat6 gene family. Infect Immun 2002; 70:5446-5453.
18.Skjot RLV, Oettinger T, Rosenkrands I, Ravn P, Brock I, Jacobsen S, et al. Comparative evaluation of low molecular mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T cell antigens. Infect Immun 2000; 68:214-220.
19.Dietrich J, Aagaard C, Leah R, Olsen AW, Stryhn A, Doherty TM, et al. Exchanging ESAT-6 with TB10.4 in an Ag85B fusion molecule-based tuberculosis subunit vaccine: efficient protection and ESAT-6 based sensitive monitoring of vaccine efficacy. J Immunol 2005; 174:6332-6339.
20.Majlessi L, Rojas MJ, Priscille B, Leclerc C. CD8+ T-cell responces of mycobacterium infected mice to a newly identified major histocompatibility complex class I -restricted epitope shared by proteins of the ESAT-6 family. Infect Immun 2003; 71:7173-7177.
21. Hervas-Stubbs S, Majlessi L, Simsova M, Morova J, Rojas MJ, Nouze C, et al. High frequency of CD4+ T- cells specific for the TB10.4 protein correlates with protection against Mycobacterium tuberculosis infection. Infect Immun 2006; 74:3396-3407.
22.Johnson BH, Hecht MH. Recombinant proteins can be isolated from Escherichia coli cells by repeated cycles of freezing and thawing. Biotechnology 1994; 12:1357-1360.