Isolation, cloning and molecular analysis of ag85a and tb10.4 genes from Mycobacterium tuberculosis
Archives of Medical Laboratory Sciences,
Vol. 3 No. 1 (2017),
16 October 2017
https://doi.org/10.22037/amls.v3i1.18317
Abstract
Background: Novel tuberculosis (TB) vaccines that aim to boost and/or replace Bacillus Calmette-Guerin (BCG) are currently in development. DNA vaccines can stimulate both humoral and cell-mediated immunity in different animal models of TB and is thought to be a promising strategy in the development of new vaccines against TB. The aim of this study was to design and construct a DNA vaccine encoding ag85a and tb10.4 fusion genes of Mycobacterium tuberculosis.
Materials and Methods: tb10.4 fragment was amplified by PCR and the product was digested with restriction
enzymes. Next, it was cloned into the pcDNA3.1+ plasmid. The ag85a gene and pcDNA3.1+/tb10.4 plasmid were digested by EcoRI and BamHI restriction enzymes. Constructed vector was sequenced. The molecular analysis was done using bioinformatics software. New chimeric vector containing ag85a-tb10.4 genes were purified. Expression of pcDNA3.1+/tb10.4-ag85a plasmid was confirmed in eukaryotic cells.
Results: Fragments of 297 bp for tb10.4 and 1017 bp for ag85a were observed in agarose gel electrophoresis.
Alignment of ag85a-tb10.4 genome sequence with reference genes in GenBank showed exact identities that indicate correction of all cloning procedures. Transfection of eukaryotic cells with pcDNA3.1+/tb10.4-ag85a vector and existence of tb10.4-ag85a fusion gene were both confirmed with RT-PCR.
Conclusion: In this study, tb10.4 and ag85a genes were isolated from Mycobacterium tuberculosis H37Rv strain and cloned into pcDNA3.1+. Also, the capability of constructed vector in producing fusion ag85a-tb10.4 protein was confirmed with RT-PCR. pcDNA3.1+/tb10.4-ag85a vector can be used for further studies in future.
- Mycobacterium tuberculosis
- ag85a
- tb10.4
- pcDNA3.1
- DNA vaccine.
How to Cite
References
Skjøt RLV, Brock I, Arend SM, Munk ME, Theisen M, Ottenhoff TH, 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.
Infection and immunity. 2002;70(10):5446-53.
Akhavan R, Meshkat Z, Khajekaramadini M, Meshkat M. Eightyear study of mycobacterium tuberculosis in mashhad, northeast of
iran. Iranian Journal of Pathology. 2013;8(2):73-80.
Asnaashari AMH, Sadrizadeh A, Ahmadi H, Meshkat M, Gholoobi A, Talab FR, et al. The study of Mycobacterium tuberculosis in Iranian patients with lung cancer. Jundishapur J
Microbiol. 2013;6:237-41.
McShane H. Tuberculosis vaccines: beyond bacille Calmette– Guérin. Philosophical Transactions of the Royal Society B: Biological Sciences. 2011;366(1579):2782-9.
Organization WH. Global tuberculosis control: WHO report 2010: World Health Organization; 2010.
Stanley SJ, Howland C, Stone MM, Sutherland I. BCG vaccination of children against leprosy in Uganda: final results. Journal of Hygiene. 1981;87(02):233-48.
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 Journal of Microbiology. 2014;7(2).
Colditz GA, Brewer TF, Berkey CS, Wilson ME, Burdick E,Fineberg HV, et al. Efficacy of BCG vaccine in the prevention of
tuberculosis: meta-analysis of the published literature. Jama.1994;271(9):698-702.
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< i>
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. European journal of immunology.
;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. The Journal of Immunology. 2006;177(9):6361-9.
Huygen K. Plasmid DNA vaccination. Microbes and infection. 2005;7(5):932-8.
Meshkat Z, Soleimanjahi H, Mahmoudi M, Hassan ZM, Mirshahabi H, Meshkat M, et al. CTL responses to a DNA vaccine
encoding E7 gene of human papillomavirus type 16 from an Iranian isolate. Iran J Immunol. 2008;5(2):82-91.
Nabavinia MS, Nasab MN, Meshkat Z, Derakhshan M, Khaje- Karamadini M. Construction and evaluation of an expression vector
containing Mtb32C (Rv0125) of Mycobacterium tuberculosis. Avicenna Journal of Medical Biotechnology. 2011;3(4):207.
Nabavinia MS, Nasab MN, Meshkat Z, Derakhshan M, Khaje- Karamadini M. Construction of an Expression Vector Containing
Mtb72F of Mycobacterium tuberculosis. Cell Journal (Yakhteh).
;14(1):61.
Flynn JL, Chan J. Immunology of tuberculosis. Annual review of immunology. 2001;19(1):93-129.
Sousa AO, Mazzaccaro RJ, Russell RG, Lee FK, Turner OC, Hong S, et al. Relative contributions of distinct MHC class Idependent
cell populations in protection to tuberculosis infection in mice. Proceedings of the National Academy of Sciences.
;97(8):4204-8.
Kassa D, Ran L, Geberemeskel W, Tebeje M, Alemu A, Selase A, et al. Analysis of immune responses against a wide range of
Mycobacterium tuberculosis antigens in patients with active pulmonary tuberculosis. Clinical and Vaccine Immunology.
;19(12):1907-15.
Gupta UD, Katoch VM, McMurray DN. Current status of TB vaccines. Vaccine. 2007;25(19):3742-51.
Hoft DF. Tuberculosis vaccine development: goals, immunological design, and evaluation. The Lancet. 2008;372(9633):164-75.
Rashidian S, Teimourpour R, Meshkat Z. Designing and construction of a DNA vaccine encoding tb10.4 gene of Mycobacterium tuberculosis. Iranian Journal of Pathology. Accepted for Publication.
Brown T. Gene cloning and DNA analysis: an introduction: John Wiley & Sons; 2010.
Invitrogen. Lipofectamine™ 2000.
Woodworth JS, Shin D, Volman M, Nunes-Alves C, Fortune SM, Behar SM. Mycobacterium tuberculosis directs immunofocusing of CD8+ T cell responses despite vaccination. The Journal of Immunology. 2011;186(3):1627-37.
Davila J, McNamara LA, Yang Z. Comparison of the predicted population coverage of tuberculosis vaccine candidates Ag85BESAT-
, Ag85B-TB10.4, and Mtb72f via a bioinformatics approach. PloS one. 2012;7(7):e40882. PubMed PMID: 22815851. Pubmed
Central PMCID: 3398899.
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. Journal of Immunology Research. 2011;2011.
Fan X, Gao Q, Fu R. Differential immunogenicity and protective efficacy of DNA vaccines expressing proteins of< i> Mycobacterium tuberculosis in a mouse model. Microbiological research. 2009;164(4):374-82.
Kaufmann SH. Future vaccination strategies against tuberculosis:thinking outside the box. Immunity. 2010;33(4):567-77.
Kremer L, Baulard A, Estaquier J, Capron A, Locht C. Analysis of the Mycobacterium tuberculosis 85A antigen promoter region. Journal of bacteriology. 1995;177(3):642-53.
Huygen K, Denis O, Montgomery DL, Yawman AM, Deck RR, DeWitt CM, et al. Immunogenicity and protective efficacy of a
tuberculosis DNA vaccine. Nature medicine. 1996;2(8):893-8.
Beyene D, Franken KL, Yamuah L, Aseffa A, Wiker HG, Kolk A, et al. Serodiagnosis of tuberculous lymphadenitis using a
combination of antigens. The Journal of Infection in Developing Countries. 2010;4(02):096-102.
Macedo GC, Bozzi A, Weinreich HR, Bafica A, Teixeira HC, Oliveira SC. Human T cell and antibody-mediated responses to the
Mycobacterium tuberculosis recombinant 85A, 85B, and ESAT-6 antigens. Journal of Immunology Research. 2010;2011.
Young D, Dye C. The development and impact of tuberculosis vaccines. Cell. 2006;124(4):683-7.
- Abstract Viewed: 426 times
- PDF Downloaded: 379 times