Document Type: Original Research
Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
Background: Tuberculosis (TB) remains as a major cause of death around the world. Construction of a new vaccine against tuberculosis is an effective way to control it. Several vaccines against this disease have been developed. The aim of the present study was to cloning of tb10.4 gene in pcDNA3.1+ plasmid and evaluation of its expression in eukaryotic cells.
Methods: Firstly, tb10.4 fragment was amplified by PCR and the PCR product was digested with restriction enzymes. Next, it was cloned into pcDNA3.1+ plasmid. Following that, pcDNA3.1+/tb10.4 recombinant plasmid was transfected into eukaryotic cells.
Results: 5700 bp band for pcDNA3.1+/tb10.4 recombinant plasmid and 297 bp fragment for tb10.4 were observed. Cloning and transfection were successful and designed recombinant vector was confirmed by sequencing.
Conclusion: Successful cloning provides a basis for the development of new DNA vaccines against tuberculosis. In the current study, the aim was cloning of tb10.4 gene in pcDNA3.1+ plasmid and transfection into eukaryotic cells.
- 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 esat-6 Gene Family. Infect Immun 2002;70(10):5446-53.
- 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(33):4412-23.
- 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(9):2455-9.
- 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(9):6361-9.
- Tang D-c, DeVit M, Johnston SA. Genetic immunization is a simple method for eliciting an immune response. Nature 1992;356(6365):152-4.
- Huygen K. Plasmid DNA vaccination. Microbes Infect 2005;7(5-6):932-8.
- Baldwin S, D'souza C, Orme I, Liu M, Huygen K, Denis O, et al. Immunogenicity and protective efficacy of DNA vaccines encoding secreted and non-secreted forms of Mycobacterium tuberculosis Ag85A. Tubercle Lung Dis 1999;79(4):251-9.
- Lu J, Wang C, Zhou Z, Zhang Y, Cao T, Shi C, et al. Immunogenicity and protective efficacy against murine tuberculosis of a prime-boost regimen with BCG and a DNA vaccine expressing ESAT-6 and Ag85A fusion protein. Clin Dev Immunol 2011;2011:617892.
- Carlotta Montagnani EC, Luisa Galli, Maurizio de Martino. Vaccine against tuberculosis: what’s new? BMC Infect Dis 2014;14(Suppl 1):1471-2334.
- Mu J, Jeyanathan M, Small CL, Zhang X, Roediger E, Feng X, et al. Immunization with a bivalent adenovirus-vectored tuberculosis vaccine provides markedly improved protection over its monovalent counterpart against pulmonary tuberculosis. Mol Ther 2009;17(6):1093-100.
- Kate W. Current Protocols in Molecular Biology. 1997:2.4.1-2.4.5. Online ISBN: 9780471142720. DOI: 10.1002/0471142727
- Brown T. Gene cloning and DNA analysis an introduction. Oxford United Kingdown: Blackwell. 2006.
- Fan X, Gao Q, Fu R. Differential immunogenicity and protective efficacy of DNA vaccines expressing proteins of Mycobacterium tuberculosis in a mouse model. Microbiol Res 2009;164(4):374-82.
- 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(4):547-54.
- Chen X OZ, Xie XL, Xu ZZ, Jiao XA. Preparation of monoclonal antibodies against mycobacterium tuberculosis TB10.4 antigen. Monoclon Antib Immunodiagn Immunother 2014 Dec;33(6):444-7.
- Sassetti CM, Boyd DH, Rubin EJ. Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 2003;48(1):77-84.
- 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(6):3396-407.
- Dietrich J, Aagaard C, Leah R, Olsen AW, Stryhn A, Doherty TM, et al. 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(10):6332-9.
- Truc Hoang CA, Jes Dietrich, Joseph P. Cassidy, Gregory Dolganov, Gary K. Schoolnik, Carina Vingsbo Lundberg, Else Marie Agger, Peter Andersen. ESAT-6 (EsxA) and TB10.4 (EsxH) Based Vaccines for Pre- and Post-Exposure Tuberculosis Vaccination. Plos One 2013 Dec;8(12):e80579.
- Ilghari D, Lightbody KL, Veverka V, Waters LC, Muskett FW, Renshaw PS, et al. Solution structure of the Mycobacterium tuberculosis EsxG.EsxH complex: functional implications and comparisons with other M. tuberculosis Esx family complexes. J Biol Chem 2011;286(34):29993-30002.
- Desta Kassa LR, Wudneh Geberemeskel, Mekashaw Tebeje et al. Analysis of Immune Responses against a Wide Range of Mycobacterium tuberculosis Antigens in Patients with Active Pulmonary Tuberculosis. Clin Vaccine Immunol 2012 Dec;19(12):1907–15.
- MacMicking JD, Taylor GA, McKinney JD. Immune control of tuberculosis by IFN-γ-inducible LRG-47 .Science 2003;302(5645):654-9.
- Vandal OH, Pierini LM, Schnappinger D, Nathan CF, Ehrt S. A membrane protein preserves intrabacterial pH in intraphagosomal Mycobacterium tuberculosis. Nat Med 2008;14(8):849-54.
- Tessema M, Koets A, Rutten V, Gruys E. Bacteriology: Review paratuberculosis: How does mycobacterium avium subsp. Paratuberculosis resist intracellular degradation? Vet Quarter 2001;23(4):153-62.
- Skjøt 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. Inf Immunol 2000;68(1):214-20.
- Palomino JC LS, Ritacco V. Tuberculosis from basic science to patient care. Belgium, Brazil, Argentina eBook 2007:93-189.
- Young D, Dye C. The development and impact of tuberculosis vaccines. Cell 2006;124(4):683-7.
- Gupta UD, Katoch VM, McMurray DN. Current status of TB vaccines. Vaccine 2007;25(19):3742-51.
- Brennan MJ. The tuberculosis vaccine challenge. Tuberculosis (Edinb) 2005;85(1-2):7-12.