Evaluation of the eukaryotic expression of mtb32C-hbha fusion gene of Mycobacterium tuberculosis in Hepatocarcinoma cell line
Abstract
Background and Objectives: HBHA and Mtb32C have been isolated from culture supernatants of Mycobacterium tu- berculosis (M. tuberculosis) and Mycobacterium bovis (M. bovis) and their immunogenicity previously studies have been confirmed. In this study, capability of constructed vector containing two mycobacterial immunodaminant antigens (Mt- b32C-HBHA), in producing new chimeric protein under the in-vitro condition was examined.
Materials and Methods: In present study Huh7.5 cells was transfected with Mtb32C-HBHA –pCDNA3.1+ recombinant vector using the calcium phosphate method and expression of chimeric protein was assessed by RT-PCR and Western blot methods.
Results: Results of RT-PCR and Western blot showed expression of 35.5 KD recombinant protein (Mtb32C-HBHA) in thiscell line.
Conclusion: The constructed vector can produce two highly immunogenic antigens that fusion of them to gather makes chi- meric antigen with new traits. Other attempts are needed to evaluate specific properties of this new antigen such as molecular conformation modeling and immunologic characteristics in future studies.
Akhavan R, Meshkat Z, Khajekaramadini M, Meshkat M. Eight-year study of Mycobacterium tuberculosis in mashhad, northeast of iran. Iran J Pathol 2013;8:73-80.
Mandell D, and Bennett. Principles and Practice of In- fectious Diseases. 8 ed: Saunders; 2014.
World Health Organization. Global tuberculosis report 2014. Available from: www.who.int/tb.
Brian WM. Topley and Wilson's Microbiology and Mi-crobial Infections. 10 ed. London: wiley; 2005.
Pym AS, Brodin P, Brosch R, Huerre M, Cole ST. Loss of RD1 contributed to the attenuation of the live tuber- culosis vaccines Mycobacterium bovis BCG and Myco- bacterium microti. Mol Microbiol 2002;46:709-717.
Dara M, Dadu A, Kremer K, Zaleskis R, Kluge HH.Epidemiology of tuberculosis in WHO European Re- gion and public health response. Eur Spine J 2013;22:549-555.
Guerrero GG, Locht C. Recombinant HBHA boosting effect on BCG-induced immunity against Mycobacte- rium tuberculosis infection. Clin Dev Immunol 2011;2011:730702.
Locht C, Hougardy JM, Rouanet C, Place S, Mascart F.Heparin-binding hemagglutinin, from an extrapulmo- nary dissemination factor to a powerful diagnostic and protective antigen against tuberculosis. Tuberculosis 2006;86(3-4):303-309.
Menozzi FD, Reddy VM, Cayet D, Raze D, Debrie AS, Dehouck MP, et al. Mycobacterium tuberculosis hepa- rin-binding haemagglutinin adhesin (HBHA) triggers receptor-mediated transcytosis without altering the in- tegrity of tight junctions. Microbes Infect 2006;8:1-9.
Guerrero GG, Feunou FP, Locht C. The coiled-coil N-terminal domain of the heparin-binding haemagglu- tinin is required for the humoral and cellular immune responses in mice. Mol Immunol 2008;46:116-124.
Pethe K, Aumercier M, Fort E, Gatot C, Locht C, Me- nozzi FD. Characterization of the heparin-binding site of the mycobacterial heparin-binding hemagglutinin adhesin. J Biol Chem 2000 12;275:14273-14280.
Masungi C, Temmerman S, Van Vooren JP, Drowart A, Pethe K, Menozzi FD. Differential T and B cell responses against Mycobacterium tuberculosis hepa- rin-binding hemagglutinin adhesin in infected healthy individuals and patients with tuberculosis. J Infect Dis 2002;185:513-520.
Temmerman ST, Place S, Debrie AS, Locht C, Mas- cart F. Effector functions of heparin-binding hemag- glutinin-specific CD8+ T lymphocytes in latent human tuberculosis. J Infect Dis 2005;192:226-232.
Ahn SS, Jeon BY, Kim KS, Kwack JY, Lee EG, Park KS, et al. Mtb32 is a promising tuberculosis antigen for DNA vaccination in pre- and post-exposure mouse models. Gene Ther 2012;19:570-575.
Ahn SS, Jeon BY, Park SJ, Choi DH, Ku SH, Cho SN, et al. Nonlytic Fc-fused IL-7 synergizes with Mtb32 DNA vaccine to enhance antigen-specific T cell re- sponses in a therapeutic model of tuberculosis. Vaccine 2013;31:2884-2890.
Begum D, Umemura M, Yahagi A, Okamoto Y, Hamada S, Oshiro K, et al. Accelerated induction of mycobacterial antigen-specific CD8+ T cells in the Mycobacterium tuberculosis-infected lung by subcu- taneous vaccination with Mycobacterium bovis bacille Calmette-Guerin. Immunology 2009;128:556-563.
Nabavinia MS, Nasab MN, Meshkat Z, Derakhshan M, Khaje-Karamadini M. Construction and evaluation of an expression vector containing Mtb32C (Rv0125) of Mycobacterium tuberculosis. AJMB 2011;3:207.
Nabavinia MS, Nasab MN, Meshkat Z, Derakhshan M, Khaje-Karamadini M. Construction of an Expression Vector Containing Mtb72F of Mycobacterium tubercu- losis. Cell J 2012;14:61-66.
Teimourpour R, Sadeghian A, Meshkat Z, Esmaelizad M, Sankian M, Jabbari AR. Construction of a DNA Vaccine Encoding Mtb32C and HBHA Genes of My- cobacterium tuberculosis. Jundishapur J Microbiol 2015;8:e21556.
Mirshahabi H, Soleimanjahi H, Pourpak Z, Meshkat Z, Hassan ZM. Production of human papilloma virus type 16 E6 oncoprotein as a recombinant protein in eukary- otic cells. Iran J Cancer Prev 2012;5:16-20.
Baghani A, Youssefi M, Safdari H, Teimourpour R, Meshkat Z. Designing and construction pcDNA3.1 vector encoding cfp10 gene of Mycobacterium tuber- culosis Jundishapur. J Microbiol 2015;8(10):e23560.
Teimourpour R, Meshkat Z, Gholoubi A, Nomani H, Rostami S. Viral load analysis of hepatitis C virus in Huh7.5 cell culture system. Jundishapur J Microbiol 2015;8(5):e19279. Epub 2015-05-31.
Meshkat Z, Soleimanjahi H, Mirshahabi H, Meshkat M, Kheirandish M, Hassan ZM. Strong immune re- sponses induced by a DNA vaccine containing HPV16 truncated E7 C-terminal linked to HSP70 gene. Iran J Immunol 2011;8:65-75.
Sambrook J. F, E. F. & Maniatis, T. Molecular Cloning:a Laboratory Manual. 2 ed: Cold Spring Harbor; 1989.
Zaman K. Tuberculosis: A Global Health Problem. J Health Popul Nutr 2010;28:111–113.
Ottenhoff TH, Kaufmann SH. Vaccines against tu- berculosis: where are we and where do we need to go? PLoS Pathog 2012;8:e1002607.
Mori T. [A new horizon of tuberculosis control and re-search]. Nihon Rinsho. 2003;61:153-159.
Orme IM, Hawkridge A. Current progress in tubercu- losis vaccine development clinical studies of TB vac- cines. Vaccine 2005;23(17-18):2105-2108.
Mueller-Ortiz SL, Sepulveda E, Olsen MR, Jagan- nath C, Wanger AR, Norris SJ. Decreased infectivity despite unaltered C3 binding by a DeltahbhA mu- tant of Mycobacterium tuberculosis. Infect Immun 2002;70(12):6751-6760.
Krishnan N, Robertson BD, Thwaites G. The mecha- nisms and consequences of the extra-pulmonary dis- semination of Mycobacterium tuberculosis. Tuberculo- sis 2010;90:361-366.
Parra M, Pickett T, Delogu G, Dheenadhayalan V, Debrie AS, Locht C, et al. The mycobacterial hepa- rin-binding hemagglutinin is a protective antigen in the mouse aerosol challenge model of tuberculosis. Infect Immun 2004;72:6799-805.
Pethe K, Alonso S, Biet F, Delogu G, Brennan MJ, Locht C, et al. The heparin-binding haemagglutinin of M. tuberculosis is required for extrapulmonary dissem- ination. Nature 2001;412(6843):190-194.
Hougardy JM, Schepers K, Place S, Drowart A, Leche- vin V, Verscheure V, et al. Heparin-binding-hemagglu-tinin-induced IFN-gamma release as a diagnostic tool for latent tuberculosis. PLoS One 2007; 2(10):e926.
Kohama H, Umemura M, Okamoto Y, Yahagi A, Goga H, Harakuni T, et al. Mucosal immunization with re- combinant heparin-binding haemagglutinin adhesin suppresses extrapulmonary dissemination of Myco- bacterium bovis bacillus Calmette-Guerin (BCG) in infected mice. Vaccine 2008;26(7):924-32. .
Skeiky YA, Alderson MR, Ovendale PJ, Guderian JA, Brandt L, Dillon DC, et al. Differential immune re- sponses and protective efficacy induced by components of a tuberculosis polyprotein vaccine, Mtb72F, deliv- ered as naked DNA or recombinant protein. J Immunol 2004;172:7618-628.
McNamara LA, He Y, Yang Z. Using epitope predic- tions to evaluate efficacy and population coverage of the Mtb72f vaccine for tuberculosis. BMC Immunol 2010;11:18.
Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL. Direct gene transfer into mouse muscle in vivo. Science 1990;23:1465-8.
Gurunathan S, Klinman DM, Seder RA. DNA vac- cines: immunology, application, and optimization. Annu Rev Immunol 2000;18:927-974.
K H. On the use of DNA vaccines for the prophylaxis of mycobacterial diseases. Infect Immun 2003;71:1613-1621.
Plotkin SA. Vaccines: the Fourth Century. Clin Vaccine Immunol 2009;16:1709–1719.
Tascon RE, Colston MJ, Ragno S, Stavropoulos E, Gregory D, Lowrie DB. Vaccination against tubercu- losis by DNA injection. Nat Med 1996;2(8):888-92.
Bruffaerts N, Huygen K, Romano M. DNA vac- cines against tuberculosis. Expert Opin Biol Ther 2014;14:1801-1813.
Lowrie DB. DNA vaccines for therapy of tuberculosis: Where are we now? Vaccine 2006;24(12):1983–9.
Romano M HK. DNA vaccines against mycobacterial diseases. Expert Rev Vaccines 2009;8:1237-1250.
| Files | ||
| Issue | Vol 8 No 2 (2016) | |
| Section | Articles | |
| Keywords | ||
| Mycobacterium tuberculosis HBHA Mtb32C Huh7.5 cells | ||
| Rights and permissions | |
|
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
