Designing and construction a DNA vaccine encoding the fusion fragment of cfp10 and Ag85A immunodominant genes of Mycobacterium tuberculosis
Archives of Medical Laboratory Sciences,
Vol. 2 No. 4 (2016),
Background: Pathogenic mycobacteria are one of major causes of human morbidity and mortality. Mycobacterium tuberculosis (M. tuberculosis) is an etiological agent of human tuberculosis. Designing new vaccines including DNA vaccines may be considered as new approaches for preventing of TB.
Materials and Methods: M. tuberculosis H37Rv was grown on Lowenstein Jensen medium for 4 weeks at 37ºC and then DNA was extracted. The cfp10 gene was amplified by PCR. After digesting the PCR product and the plasmid, cfp10 fragment was ligated into the vector using T4 DNA ligase. Then, Ag85A was subcloned into pcDNA/cfp10. Escherichia coli strain JM109 bacteria were transformed by the desired construct. Clone confirmations were performed by colony PCR, restriction enzyme digestion and DNA sequencing. Recombinant vector was transfected into HeLa cells and total RNA was extracted, then cDNA was synthesized using oligo-dT. Finally PCR was performed by cfp10 primers.
Results: The cfp10 was amplified by PCR method and the PCR products were visualized by agarose gel electrophoresis. The cfp10 fragments showed 303 bp in length. The cfp10 cloned into pcDNA. Then, Ag85Awas ligated into pcDNA/cfp10 after digestion correctly. Colony-PCR and restriction enzyme digestion and sequencing confirmed the cloning the fusion Ag85A/cfp10 fragment. Finally, after cDNA synthesis, expression of vector was confirmed in eukaryotic system.
Conclusion: Cloning of Ag85A/cfp10 genes of M. tuberculosis were performed correctly. It can use as a DNA vaccine for investigation the immune responses in animal models in future studies.
- DNA vaccine
- Mycobacterium tuberculosis
How to Cite
Akhavan R, Meshkat Z, karamadini MK, Meshkat M. Eight-year Study of Mycobacterium tuberculosis in Mashhad, Northeast of Iran. Iranian Journal of Pathology. 2013;8(2):73.
Amir Mohamad HA, Ali S, Hamid A, Mojtaba M, Aida G, Fariba Rezai T, et al. The Study of Mycobacterium tuberculosis in Iranian Patients With Lung Cancer. Jundishapur Journal of Microbiology. 2013;6(3):237-41.
Anandaiah A, Dheda K, Keane J, Koziel H, Moore DA, Patel NR. Novel developments in the epidemic of human immunodeficiency virus and tuberculosis coinfection. American journal of respiratory and critical care medicine. 2011;183(8):987-97.
Dey A, Kumar U, Sharma P, Singh S. Immunogenicity of candidate chimeric DNA vaccine against tuberculosis and leishmaniasis. Vaccine. 2009;27(37):5152-60.
Nabavinia MS, Naderi Nasab M, Meshkat Z, Derakhshan M, Khaje-Karamadini M. Construction of an Expression Vector Containing Mtb72F of Mycobacterium tuberculosis. Cell J. 2012Spring;14(1):61-6. PubMed PMID: 23626939. Pubmed Central PMCID: 3635822. Epub 2013/04/30. eng.
Okada M. Novel vaccines against M. tuberculosis. Kekkaku. 2006 Dec;81(12):745-51. PubMed PMID: 17240920. Epub 2007/01/24. jpn.
Orme IM. Preclinical testing of new vaccines for tuberculosis: A comprehensive review. Vaccine. 2006;24(1):2-19.
Teimourpour R, Sadeghian A, Meshkat Z, Esmaelizad M, Sankian M, Jabbari A-R. Construction of a DNA Vaccine Encoding Mtb32C and HBHA Genes of Mycobacterium tuberculosis. Jundishapur journal of microbiology. 2015;8(8).
Malin AS, Huygen K, Content J, Mackett M, Brandt L, Andersen P, et al. Vaccinia expression of Mycobacterium tuberculosis-secreted proteins: tissue plasminogen activator signal sequence enhances expression and immunogenicity of M. tuberculosis Ag85. Microbes and Infection. 2000;2(14):1677-85.
Nabavinia MS, Nasab Mn Fau - Meshkat Z, Meshkat Z Fau - Derakhshan M, Derakhshan M Fau - Khaje-Karamadini M, Khaje-Karamadini M. Construction and Evaluation of an Expression Vector Containing Mtb32C (Rv0125) of Mycobacterium tuberculosis. Avicenna J Med Biotechnol. 2011;3(4):207-10. eng.
Flint JL, Kowalski JC, Karnati PK, Derbyshire KM. The RD1 virulence locus of Mycobacterium tuberculosis regulates DNA transfer in Mycobacterium smegmatis. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(34):12598-603.
Romano M, D'Souza S, Adnet P-Y, Laali R, Jurion F, Palfliet K, et al. Priming but not boosting with plasmid DNA encoding mycolyl-transferase Ag85A from Mycobacterium tuberculosis increases the survival time of Mycobacterium bovis BCG vaccinated mice against low dose intravenous challenge with M. tuberculosis H37Rv. Vaccine. 2006;24(16):3353-64.
Gupta UD, Katoch VM, McMurray DN. Current status of TB vaccines. Vaccine. 2007;25(19):3742-51.
Berthet FX, Rasmussen PB, Rosenkrands I, Andersen P, Gicquel B. A Mycobacterium tuberculosis operon encoding ESAT-6 and a novel low-molecular-mass culture filtrate protein (CFP-10). Microbiology. 1998;144(11):3195-203.
Guo S, Xue R, Li Y, Wang SM, Ren L, Xu JJ. The CFP10/ESAT6 complex of Mycobacterium tuberculosis may function as a regulator of macrophage cell death at different stages of tuberculosis infection. Medical Hypotheses. 2012;78(3):389-92.
Denis O, Tanghe A, Palfliet K, Jurion F, Van Den Berg TP, Vanonckelen A, et al. Vaccination with Plasmid DNA Encoding Mycobacterial Antigen 85A Stimulates a CD4+ and CD8+ T-Cell Epitopic Repertoire Broader than That Stimulated byMycobacterium tuberculosis H37Rv Infection. Infection and immunity. 1998;66(4):1527-33.
Nakano H, Nagata T, Suda T, Tanaka T, Aoshi T, Uchijima M, et al. Immunization with dendritic cells retrovirally transduced with mycobacterial antigen 85A gene elicits the specific cellular immunity including cytotoxic T-lymphocyte activity specific to an epitope on antigen 85A. Vaccine. 2006;24(12):2110-9.
Lee BY, Horwitz MA. T-cell epitope mapping of the three most abundant extracellular proteins of Mycobacterium tuberculosis in outbred guinea pigs. Infection and immunity. 1999;67(5):2665-70.
Grover A, Ahmed MF, Singh B, Verma I, Sharma P, Khuller GK. A multivalent combination of experimental antituberculosis DNA vaccines based on Ag85B and regions of difference antigens. Microbes Infect. 2006 Aug;8(9-10):2390-9. PubMed PMID: 16962360. Epub 2006/09/12. eng.
Baghani A, Youssefi M, Safdari H, Teimourpour R, Meshkat Z. Designing and Construction Pcdna3. 1 Vector Encoding Cfp10 Gene of Mycobacterium tuberculosis. Jundishapur Journal of Microbiology. 2015;8(10).
Tyagi AK, Nangpal P, Satchidanandam V. Development of vaccines against tuberculosis. Tuberculosis. 2011;91(5):469-78.
Meshkat Z, Mirshahabi H, Soleimanjahi H, Mohamad Hassan Z. Construction a DNA Vaccine Containing Human Papillomavirus Type 16 Early Genes as a Potential Vaccine for Cervical Cancer Prevention and Therapy. Iranian Journal of Pathology. 2009;4(2):65-70.
Bao H, Yu T, Jin Y, Teng C, Liu X, Li Y. Construction of a DNA vaccine based on the Mycobacterium tuberculosis Ag85A/MPT64 fusion gene and evaluation of its immunogenicity. Molecular Medicine Reports. 2012;6(6):1375-8.
Launois P, DeLeys R, Niang M, Drowart A, Andrien M, Dierckx P, et al. T-cell-epitope mapping of the major secreted mycobacterial antigen Ag85A in tuberculosis and leprosy. Infection and immunity. 1994;62(9):3679-87.
Lozes E, Huygen K, Content J, Denis O, Montgomery DL, Yawman AM, et al. Immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex. Vaccine. 1997;15(8):830-3.
Gao H, Yue Y, Hu L, Xu W, Xiong S. A novel DNA vaccine containing multiple TB-specific epitopes casted in a natural structure (ECANS) confers protective immunity against pulmonary mycobacterial challenge. Vaccine. 2009;27(39):5313-9.
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. Clinical and Developmental Immunology. 2010;2011.
- Abstract Viewed: 818 times
- PDF Downloaded: 395 times