Showing 10 results for Mycobacterium Tuberculosis
Bagher Moradi, Mojtaba Sankian, Yousef Amini, Zahra Meshkat,
Volume 4, Issue 2 (5-2016)
Abstract
Background: Mycobacterium tuberculosis is the causative agent of tuberculosis (TB). Bacille Calmette-Guerin (BCG) vaccine, is not effective in adults, therefore, many efforts have been made to produce an effective adult TB vaccine. The aim of this study was to develop a new tuberculosis DNA vaccine candidate encoding a recombinant HspX-PPE44-EsxV fusion antigen of M. tuberculosis.
Methods: A fusion DNA segment consisting of HspX, linker, PPE44, linker, and EsxV, after codon optimization, was designed. The fusion DNA was cloned and its sequence confirmed. Then, expression of a recombinant pcDNA3.1 (+)/HspX-PPE44-EsxV plasmid in Chinese hamster ovary (CHO) cells was verified by RT-PCR and Western-blot analysis.
Results: A 1968 bp band in RT-PCR and a 68 kDa band on Western-blot analysis confirmed transcription and expression of recombinant hspX-ppe44-esxV in eukaryotic cells.
Conclusions: A recombinant DNA segment encoding the HspX-PPE44-EsxV fusion antigen of M. tuberculosis was constructed and considered to be tested as a new TB DNA vaccine candidate.
Atieh Yaghoubi, Ehsan Aryan, Hosna Zare, Shadi Alami, Roghayeh Teimourpour, Zahra Meshkat,
Volume 5, Issue 1 (10-2016)
Abstract
Background: Tuberculosis (TB) is a major cause of death worldwide. Finding an effective vaccine against TB is the best way to control it. Several vaccines against this disease have been developed but none are completely protective. The aim of this study was to design and construct a cloning vector containing the Mycobacterium tuberculosis (M. tuberculosis) heat shock protein X (hspX).
Methods: First, an hspX fragment was amplified by PCR and cloned into plasmid pcDNA3.1(+) and recombinant vector was confirmed.
Results: A 435 bp hspX fragment was isolated. The fragment was 100% homologous with hspX of M. tuberculosis strain H37Rv in GenBank.
Conclusions: In this study, the cloning vector pcDNA3.1(+), containing a 435-bp hspX fragment of M. tuberculosis, was constructed. This could be used as a DNA vaccine to induce immune responses in animal models in future studies.
Farzad Khademi, Arshid Yousefi-Avarvand, Mohammad Derakhshan, Zahra Meshkat, Mohsen Tafaghodi, Kiarash Ghazvini, Ehsan Aryan, Mojtaba Sankian,
Volume 6, Issue 1 (10-2017)
Abstract
Background: The purpose of this study was to clone, express, and purify a novel multidomain fusion protein of Micobacterium tuberculosis (Mtb) in a prokaryotic system.
Methods: An hspX/esxS gene construct was synthesized and ligated into a pGH plasmid, E. coli TOP10 cells were transformed, and the vector was purified. The vector containing the construct and pET-21b (+) plasmid were digested with the same enzymes and the construct was ligated into pET-21b (+). The accuracy of cloning was confirmed by colony PCR and sequencing. E. coli BL21 cells were transformed with the pET-21b (+)/hspX/esxS expression vector and protein expression was evaluated. Finally, the expressed fusion protein was purified on a Ni-IDA column and verified by SDS-PAGE and western blotting.
Results: The hspX/esxS gene construct was inserted into pET-21b (+) and recombinant protein expression was induced with IPTG in E. coli BL21 cells. Various concentrations of IPTG were tested to determine the optimum concentration for expression induction. The recombinant protein was expressed in insoluble inclusion bodies. Three molar guanidine HCl was used to solubilize the insoluble protein.
Conclusions: An HspX/EsxS Mtb fusion protein was expressed in E. coli and the recombinant protein was purified. After immunological analysis, the HspX/EsxS fusion protein might be an anti-tuberculosis vaccine candidate in future clinical trial studies.
Maryam Sadat Nabavinia, Mohammad Ramezani, Aida Gholoobi, Mahboubeh Naderinasab, Zahra Meshkat,
Volume 6, Issue 1 (10-2017)
Abstract
Background: With one-third of the world’s population infected, tuberculosis (TB) is one of the most common infectious diseases and a major public health problem, especially in developing countries. The efficacy of the BCG vaccine for controlling the disease in adults is poor. The development of an effective TB vaccine is a global objective. An effective tuberculosis vaccine should stimulate cellular immunity. DNA vaccines are a new generation of vaccines with the potential to achieve this goal. The aim of this study was to produce a DNA vaccine of Mtb72F.
Methods: mtb32C, mtb39, and mtb32N were cloned into pcDNA3.1 using restriction enzyme digestion and T4 DNA ligase. Colony-PCR and restriction enzyme digestion were performed to detect transformed bacteria. DNA sequencing confirmed the desired gene insertion into the vector. A Chinese hamster ovary (CHO) cell line was transfected with the recombinant plasmid and RT-PCR was performed to assess gene expression.
Results: Gel electrophoresis showed the expected amplified gene fragments of 429, 614, and 1200 base pairs (bps) for mtb32C, mtb32N, and mtb39, respectively. Enzyme digestion and gel electrophoresis showed the expected fragments, indicating the desired gene position and orientation in the recombinant plasmid. This finding was verified by DNA sequencing, and RT-PCR demonstrated gene expression in the CHO cell line.
Conclusions: An Mtb72F DNA plasmid was successfully constructed. This plasmid may be a candidate for animal immunizations.
Yousef Amini, Mohsen Tafaghodi, Saied Amel Jamehdar, Zahra Meshkat, Bagher Moradi, Mojtaba Sankian,
Volume 6, Issue 2 (5-2018)
Abstract
Background: Subunit vaccines are appropriate vaccine candidates for the prevention of some infections. In this study, three immunogenic proteins of Mycobacterium tuberculosis, including HspX, Ppe44, and EsxV as a new construction, were expressed alone and as a fusion protein to develop a new vaccine candidate against tuberculosis infection.
Methods: To make the fusion protein, the three genes were linked together by AEAAAKEAAAKA linkers and inserted into pET21b and pET32b vectors. Escherichia coli (E. coli) Top10 cells were transformed with the plasmid, and the purified plasmid was used to transform E. coli BL21 cells. Protein expression was induced with IPTG. After optimizing protein expression, the recombinant proteins were purified by Ni-NTA chromatography. Protein purification was confirmed by SDS-PAGE and Western blotting with an anti-poly histidine-peroxidase monoclonal antibody against the 6His–tags at the proteins’ C termini.
Results: Directional cloning was confirmed by polymerase chain reaction (PCR), restriction enzyme digestion, and sequencing. The highest expression of the tri-fusion protein and HspX were obtained by the addition of 0.2 mM of IPTG to E. coli BL-21 cells at 37 ˚C and 18 h of incubation. For Ppe44 and EsxV, the optimum expression conditions were 18 ˚C and 16 h of incubation. SDS-PAGE and Western blots confirmed that the desired proteins were produced.
Conclusions: The three desired proteins and the fusion protein were successfully expressed and the conditions for optimum expression determined. These recombinant proteins will be evaluated as vaccine candidates against tuberculosis. Further studies are needed to evaluate the abilities of these proteins to induce strong immunological responses.
Mr Davood Mansury, Kiarash Ghazvini, Saeid Amel Jamehdar, Dr Ali Badiee, Mohsen Tafaghodi, Amin Reza Nikpoor, Yousef Amini, Mahmoud Reza Jaafari,
Volume 7, Issue 2 (1-2019)
Abstract
Background: Due to the ineffectiveness of the BCG vaccine, especially in adult pulmonary tuberculosis (TB), and variable efficacies against childhood forms of TB, developing an effective TB vaccine is a major priority in controlling this disease. The aim of this study was to evaluate the immunogenicity of a DOTAP liposome formulation containing a fusion protein (FP) containing Mycobacterium tuberculosis HspX, PPE44, and EsxV.
Methods: The FP was expressed in E. coli BL21 cells and confirmed by SDS-PAGE and Western blots. The FP was then encapsulated in various liposomal formulations. Afterwards, liposomal size, zeta potential, and encapsulation efficiency were evaluated. Mice were subcutaneously vaccinated on days 0, 14, and 28 with liposomes containing the FP. Two weeks after the last injection, IFN-γ, IL-4, IL-17, and IL-12 in spleen cell culture supernatants, and IgG2a, IgG1, and IgG2b titers in sera were measured.
Results: The FP concentration was 1mg/ml. The encapsulation efficiency of the liposomes varied from 69% in Lip (DOTAP/TDB/CHOL/FP) to 80% in Lip (DOTAP/CHOL/FP). The greatest IFN-γ and IL-12 levels were observed in BCG-primed mice that were boosted with Lip (DOTAP/CHOL/FP). In addition, IL-17 production was significantly greater in all groups than controls except in those that received histidine buffer and FP. The IgG2a/IgG1 ratios were greater in the Lip (DOTAP/TDB/CHOL/FP), Lip (DOTAP/CHOL/FP), Lip (DOTAP/CHOL), and BCG-primed and Lip (DOTAP/CHOL/FP)-boosted groups than in the other groups, indicating a cellular immune response.
Conclusions: The liposomes containing DOTAP combined with the fusion protein induced a Th1 response. The mice that first received BCG and then Lip (DOTAP/CHOL/FP), produced the most IFN-γ and IL-12, indicating a strong Th1 response.
Faria Hasanzadeh Haghighi, Ehsan Aryan, Aida Gholoobi, Hosna Zare, Zahra Meshkat,
Volume 8, Issue 1 (5-2019)
Abstract
Background: Tuberculosis (TB) is the leading cause of death by infectious diseases worldwide, and especially prevalent in developing countries. Several vaccines against TB have been developed, recently. The aim of the present study was to design and construct a cloning vector encoding Mycobacterium tuberculosis (MTB) mpt51 gene.
Methods: DNA was extracted from MTB H37Rv strain. Gene-specific primers were designed using Gene Runner software and the mpt51 gene was amplified by PCR. The amplified fragment and pcDNA3.1(+) cloning vector were both digested with restriction enzymes, the mpt51 fragment was ligated into the vector, and the Escherichia coli (E. coli) TOP10 strain were transformed by the recombinant plasmid. Positive clones were identified by colony PCR, restriction enzyme digestion, and DNA sequencing.
Results: The mpt51 gene was successfully cloned into pcDNA3.1(+). A 6400 bp band for the pcDNA3.1(+)/mpt51 recombinant plasmid and a 926 bp band for mpt51 were observed by colony PCR, and restriction enzyme digestion on agarose gels. The DNA sequence was 100% homologous with the mpt51 fragment of H37Rv in GenBank.
Conclusions: In the current study, the mpt51 gene of MTB was correctly cloned into pcDNA3.1(+). The expression of this recombinant vector can be studied in eukaryotic cells. Moreover, it is possible to determine the efficacy of this vector as a DNA vaccine candidate, and to test its protective function compared to BCG in animal models in future.
Mahdis Ghavidel, Keyvan Tadayon, Nader Mosavari, Kimiya Nourian, Hamid Reza Bahramitaghanaki, Gholam Reza Mohammadi, Mohammad Rashtibaf, Kiarash Ghazvini,
Volume 8, Issue 3 (10-2019)
Abstract
Background: Tuberculosis (TB) still remains endemic worldwide making epidemiological studies essential to mitigating efforts implicated in identifying its source,controlling, and preventing the spread of dangerous strains amongst humans such as Mycobacterium tuberculosis (Mtb).
Methods: In this study, we sought to determine the 6 Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat (MIRU-VNTR) loci with high discriminatory powers for Mtb genotypingas well as the loci with the highest and the lowest discriminatory powers for MIRU-VNTR.To conduct our search, we used several databases such as science direct, Embase (Elsevier), Web of Science, Scopus and Medline via PubMed. Searches were performed using key words including: Mycobacterium tuberculosis, MIRU–VNTR, Allele diversity, Genetic diversity and human patient. Finally, 56 articles were selected after filtering out titles, abstracts and full texts.
Results: Loci with high discriminatory powers included MIRU10 and MIRU26, while MIRU2, MIRU20, MIRU24 and ETRD had poor discriminatory powers. According to previous data in the literature, the loci MIRU10, MIRU26, MIRU40, QUB 26, QUB 11b and Mtub21 have high discriminatory powers.
Conclusions:Therefore, these loci recommended for genotyping Mtb to save time and cost and to ensure the production of reliable results.
Omid Salemi, Zahra Noormohammadi, Fariborz Bahrami, Seyed Davar Siadat, Soheila Ajdary,
Volume 8, Issue 4 (1-2020)
Abstract
Background: It is estimated that one third of the world's population is infected with Mycobacterium tuberculosis (Mtb), the causative agent of Tuberculosis (TB). The BCG vaccine is widely used to fight against TB; however, many question its ability to provide complete protection from Mtb. Recently, the "Region of Difference 1" (RD1) set of genes were shown to be involved in the pathogenesis of Mtb. Downstream of RD1 transcription region, two proteins are encoded, known as EspB and EspC, which were found to contribute to Mtb virulence. In this study these two proteins are targeted as potential vaccine candidates against TB.
Methods: The EspB and EspC Mtb genes were codon-optimized for expression and synthesis in Escherichia coli (E. coli). The amplicons were cloned into a pET21a expression vector and transformed into E. coli BL21(DE3). The expression and purity of the expressed proteins (i.e. rEspC, rEspB and rEspC/EspB) were confirmed by SDS-PAGE and Western blotting. Moreover, BALB/c mice were immunized against Mtb using the recombinant proteins. Finally, the mice sera were analyzed via Western blotting.
Results: EspC, EspB, and EspC/EspB fusion genes were cloned and expressed in E. coli. Both SDS-PAGE and Western blots confirmed the presence and successful purification of the desired proteins. Moreover, antisera produced against the purified recombinant proteins reacted with Mtb proteins.
Conclusions: rEspC, rEspB, and rEspC/EspB could be expressed and purified using an E. coli expression system. The recombinant proteins induced the production of antibodies in BALB/c mice that reacted with Mtb proteins.
Omid Salemi, Zahra Noormohammadi, Fariborz Bahrami, Seyed Davar Siadat, Soheila Ajdary,
Volume 11, Issue 4 (2-2023)
Abstract
Background: Two newly identified proteins, EspB and EspC are involved in the pathogenesis of Mycobacterium tuberculosis. The objective of the present study was to evaluate the immunogenicity of recombinant EspC, EspB, and EspC/EspB fusion proteins in mice.
Methods: BALB/c mice were immunized subcutaneously with recombinant EspC, EspB, and fusion EspC/EspB proteins, three times with along with Quil-A as an adjuvant. The cellular and humoral immune responses were evaluated by quantifying IFN-g, IL-4, IgG, IgG1, and IgG2a antibodies against the antigens.
Results: The results showed that the mice immunized with recombinant EspC, EspB, and EspC/EspB proteins did not produce IL-4, whereas IFN-g was secreted in response to all three proteins. EspC/EspB group produced significant amounts of IFN-g in response to stimulation with all the three recombinant proteins (P<0.001). In mice immunized with EspC, high levels of IFN-g were detected in response to EspC/EspB, and EspC (P<0.0001); while mice immunized with EspB produced lower levels of IFN- g in response to EspC/EspB, and EspB (P<0.05).
Conclusions: All the three recombinant proteins induced Th1-type immune responses in mice against EspB and EspC; however, EspC/EspB protein is more desirable due to the presence of epitopes from both EspC and EspB proteins and the production of immune responses against both.
