Design and construction of fusion genes hspX and tb10.4 from Mycobacterium tuberculosis in a cloning vector

authors:

avatar Atieh Yaghoubi , avatar Ehsan Aryan , avatar Mohammad Derakhshan , avatar Zahra Meshkat ORCID , *


how to cite: Yaghoubi A, Aryan E, Derakhshan M, Meshkat Z. Design and construction of fusion genes hspX and tb10.4 from Mycobacterium tuberculosis in a cloning vector. koomesh. 2017;19(1):e151335. 

Abstract

Introduction: Tuberculosis (TB) is the most important infectious disease and is one of the most common causes of death in the world especially in developing countries. TB is caused by infection with Mycobacterium tuberculosis. Designing and construction new vaccines against Mycobacterium tuberculosis are the only effective way to prevent and control the disease. The aim of this study was to design and construct a cloning vector encoding hspX and tb10.4 fusion genes of Mycobacterium tuberculosis. Materials and Methods: At first, tb10.4 fragment was amplified by PCR method and it was digested with restriction enzymes and was cloned into the plasmid pcDNA3.1 +. Then, hspX fragment was amplified by PCR method and it was digested by HindIII and BamHI restriction enzymes. The recombinant plasmid pcDNA3.1 + / tb10.4 also digested with the same enzymes and hspX was subcloned into the recombinant vector. This construct was transformed into the Escherichia coli strain TOP10. The confirming the clones were performed by colony PCR, restriction enzyme digestion and sequencing methods. Results: PCR and enzymatic digestion products were run on the agarose gel and tb10.4 and hspX genes were observed 291bp and 435bp, respectively. In addition, results of DNA sequencing showed 100% homology with hspX and tb10.4 genes of Mycobacterium tuberculosis H37Rv strain recorded in GenBank. Conclusion: In this study, hspX and tb10.4 genes of Mycobacterium tuberculosis were cloned into pcDNA3.1 + vector correctly. This vector can use as a DNA vaccine to induce immune system responses in animal models in future studies.

References

  • 1.

    Akhavan R, Meshkat Z, Jamehdar S. Comparing the frequency of mycobacterium tuberculosis with direct microscopy and culture methods. Jundishapur J Microbiol 2012; 6: 95-96. (Persian).

  • 2.

    Gholoobi A, Masoudi-Kazemabad A, Meshkat M, Meshkat Z. Comparison of culture and PCR methods for diagnosis of mycobacterium tuberculosis in different clinical specimens. Jundishapur J Microbiol 2014; 7: e8939. (Persian).

  • 3.

    Mohammadbeigi A, Dalirian S, Mokhtari M, Jadidi R. Delay in diagnosis and treatment of pulmonary tuberculosis and its association with some social and personal characteristics in Markazi Province (2008-2014). Koomesh 2015; 4: 966-973. (Persian).

  • 4.

    Nabavinia MS, Meshkat Z, Derakhshan M, Khaje-Karamadini M. Construction of an expression vector containing Mtb72F of mycobacterium tuberculosis. Cell J (Yakhteh) 2012; 14: 61. (Persian).

  • 5.

    Kaufmann SH. Fact and fiction in tuberculosis vaccine research: 10 years later. Lancet Infect Dis 2011; 11: 633-640.

  • 6.

    Baghani A, Youssefi M, Safdari H, Teimourpour R, Meshkat Z. Designing and construction pcdna3. 1 vector encoding Cfp10 gene of mycobacterium tuberculosis. Jundishapur J Microbiol 2015; 8. (Persian).##.

  • 7.

    Gupta UD, Katoch VM, McMurray DN. Current status of TB vaccines. Vaccine 2007; 25: 3742-3751.

  • 8.

    Colditz GA, Brewer TF, Berkey CS, Wilson ME, Burdick E, Fineberg HV. Efficacy of BCG vaccine in the prevention of tuberculosismeta-analysis of the published literature. Jama 1994; 271: 698-702.

  • 9.

    Hoang T, Aagaard C, Dietrich J, Cassidy JP, Dolganov G, Schoolnik GK, et al. ESAT-6 (EsxA) and TB10. 4 (EsxH) based vaccines for pre-and post-exposure tuberculosis vaccination. PloS One 2013; 8: e80579.

  • 10.

    Barker LF, Brennan MJ, Rosenstein PK, Sadoff JC. Tuberculosis vaccine research: the impact of immunology. Curr Opin Immunol 2009; 21: 331-338.

  • 11.

    Britton WJ, Palendira U. Improving vaccines against tuberculosis. Immunol Cell Biol 2003; 81: 34-45.

  • 12.

    Wieczorek AE, Troudt JL, Knabenbauer P, Taylor J, Pavlicek RL, Karls R, et al. HspX vaccination and role in virulence in the guinea pig model of tuberculosis. Pathog Dis 2014; 71: 315-325.

  • 13.

    Taylor JL, Wieczorek A, Keyser AR, Grover A, Flinkstrom R, Karls RK, et al. HspX-mediated protection against tuberculosis depends on its chaperoning of a mycobacterial molecule. Immunol Cell Biol 2012; 90: 945-954.

  • 14.

    Skjt RL, Oettinger T, Rosenkrands I, Ravn P, Brock I, Jacobsen S. 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.

  • 15.

    Dietrich J, Aagaard C, Leah R, Olsen AW, Stryhn A, Doherty TM. Exchanging ESAT6 with TB10. 4 in an Ag85B fusion molecule-based tuberculosis subunit vaccine: efficient protection and ESAT6-based sensitive monitoring of vaccine efficacy. J Immunol 2005; 174: 6332-6339.

  • 16.

    Baghani A, Youssefi M, Safdari H, Teimourpour R, Meshkat Z. Designing and construction pcdna3. 1 vector encoding Cfp10 gene of mycobacterium tuberculosis. Jundishapur J Microbiol 2015; 8. (Persian).

  • 17.

    Tajeddin E, Kargar M, Noroozi J, Ahmadi M, Kazempour M. Identification of mycobacterium tuberculosis beijing genotype using three different molecular methods. Koomesh 2009; 1: 7-14. (Persian).

  • 18.

    D'Souza S, Denis O, Scorza T, Nzabintwali F, Verschueren H, Huygen K. CD4+ T cells contain Mycobacterium tuberculosis infection in the absence of CD8+ T cells in mice vaccinated with DNA encoding Ag85A. Eur J Immunol 2000; 30: 2455-2459.

  • 19.

    Tanghe A, Lefvre P, Denis O, DSouza S, Braibant M, Lozes E, et al. Immunogenicity and protective efficacy of tuberculosis DNA vaccines encoding putative phosphate transport receptors. J Immunol 1999; 162: 1113-1119.

  • 20.

    Radoevi K, Wieland CW, Rodriguez A, Weverling GJ, Mintardjo R, Gillissen G, et al. Protective immune responses to a recombinant adenovirus type 35 tuberculosis vaccine in two mouse strains: CD4 and CD8 T-cell epitope mapping and role of gamma interferon. Infect Immun 2007; 75: 4105-4115.

  • 21.

    Wieczorek AE, Troudt JL, Knabenbauer P, Taylor J, Pavlicek RL, Karls R, et al. HspX vaccination and role in virulence in the guinea pig model of tuberculosis. Pathog Dis 2014; 71: 315-325.

  • 22.

    Kamath A, Woodworth JS, Behar SM. Antigen-specific CD8+ T cells and the development of central memory during Mycobacterium tuberculosis infection. J Immunol 2006; 177: 6361-6369.

  • 23.

    Sun R, Skeiky YA, Izzo A, Dheenadhayalan V, Imam Z, Penn E, et al. Novel recombinant BCG expressing perfringolysin O and the over-expression of key immunodominant antigens; pre-clinical characterization, safety and protection against challenge with Mycobacterium tuberculosis. Vaccine 2009; 27: 4412-4423.

  • 24.

    Romano M, Aryan E, Korf H, Bruffaerts N, Franken C, Ottenhoff T. Potential of mycobacterium tuberculosis resuscitation-promoting factors as antigens in novel tuberculosis sub-unit vaccines. Microbes Infect 2012; 14: 86-95.

  • 25.

    Soleimanpour S, Farsiani H, Mosavat A, Ghazvini K, Eydgahi MR, Sankian M, et al. APC targeting enhances immunogenicity of a novel multistage Fc-fusion tuberculosis vaccine in mice. Appl Microbiol Biotechnol 2015; 99: 10467-10480.

  • 26.

    Xin Q, Niu H, Li Z, Zhang G, Hu L, Wang B, et al. Subunit vaccine consisting of multi-stage antigens has high protective efficacy against mycobacterium tuberculosis Infection in Mice. PloS One 2013; 8: e72745.

  • 27.

    Marongiu L, Donini M, Toffali L, Zenaro E, Dusi S. ESAT-6 and HspX improve the effectiveness of BCG to induce human dendritic cells-dependent Th1 and NK cells activation. PloS One 2013; 8: e75684.

  • 28.

    Niu H, Hu L, Li Q, Da Z, Wang B, Tang K, et al. Construction and evaluation of a multistage Mycobacterium tuberculosis subunit vaccine candidate Mtb10. 4HspX. Vaccine 2011; 29: 9451-9458##.