scholarly journals Membrane and Secretory Protein Extraction of Mycobacterium Tuberculosis and Mycobacterium Bovis Using One Dimensional Electrophoresis (SDS-PAGE)

2017 ◽  
pp. 040-044
Author(s):  
AH Tasbiti
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Protein Extraction ◽  
Secretory Protein ◽  
One Dimensional ◽  
Sds Page ◽  
Dimensional Electrophoresis

The Protein Extraction from Longan Pulp (Dimocarpus longan Lour. cv. Daw) for Proteomic Analysis Using One-Dimensional Electrophoresis

2019 ◽  
Vol 886 ◽  
pp. 21-26 ◽  
Author(s):  
Achara Kleawkla ◽  
Ekawit Threenet ◽  
Wanlapha Khonkham ◽  
Winai Wiriyaalongkorn ◽  
Adisak Joomwong ◽  
...  
Keyword(s):  
Protein Expression ◽  
Proteomic Analysis ◽  
Protein Extraction ◽  
Protein Pattern ◽  
Elongation Factor ◽  
Fruit Growth ◽  
Physiological Disorder ◽  
One Dimensional ◽  
Dimocarpus Longan ◽  
Dimensional Electrophoresis

Three procedures for protein extraction in longan pulp had been applied to analyze protein pattern and quality of Longan pulp (Dimocarpus longan Lour. cv. Daw) during fruit growth to increase protein expression in proteomic analysis at Maejo university’s farm. There were data points to compare between normal and physiological disorder syndromes during fruit growth (5,10, 15, 20, 25 and 30 weeks, respectively) by one dimensional electrophoresis (1-D gel) technique in reducing condition. The first protein extraction, M1 (95% ethanol) showed obviously 15 protein bands which molecular weights were 14.97, 17.90, 18.30, 21.63, 28.54, 31, 33.96, 35.02, 42, 51.69, 65.69, 71.54, 88.02, 106.86 and 130 kDa, respectively. While M2 extraction (phenol-methanol/ammonium acetate) and M3 extraction (1.5 mM tris-HCl pH 8.0, 5 mM EDTA, 2% SDS) had low protein expression and no sharpness (13 and 12 protein bands, respectively). In different extraction conditions, therefore, M1 was a suitable method because of highest protein bands and obvious protein expression on longan pulp for proteomic analysis. Proteomic analysis of M1 extraction method was used in protein analysis by using LC-MS / MS techniques. It was found that the heat shock protein 83 (81.0 kDa), a family of proteins that was produced by cells in response to exposure on stressful conditions, the elongation factor 1-alpha (49.45 kDa), a selective regulator of translation, and the peroxidase 4 (39.74 kDa), a protein that is involved in the degeneration or aging of cells. These proteins exhibited a darker appearance of the protein bands at 30 weeks. Moreover, a partial glyceraldehyde-3-phosphate dehydrogenase (34.06 kDa), the protein involved in metabolic processes in glucose degradation, was also founded a darker appearance at 25 weeks and low appearance at 30 weeks of abnormal longan. However, higher proteomic techniques should be studied to confirm this biomarker protein in the further.

scholarly journals Listeria-Vectored Vaccine Expressing the Mycobacterium tuberculosis 30-Kilodalton Major Secretory Protein via the Constitutively Active prfA* Regulon Boosts Mycobacterium bovis BCG Efficacy against Tuberculosis

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Qingmei Jia ◽  
Barbara Jane Dillon ◽  
Saša Masleša-Galić ◽  
Marcus A. Horwitz
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Interleukin 2 ◽  
Secretory Protein ◽  
Broth Culture ◽  
Listeriolysin O ◽  
Content Type ◽  
Ifn Γ ◽  
Constitutively Active

ABSTRACT A potent vaccine against tuberculosis, one of the world's deadliest diseases, is needed to enhance the immunity of people worldwide, most of whom have been vaccinated with the partially effective Mycobacterium bovis BCG vaccine. Here we investigate novel live attenuated recombinant Listeria monocytogenes (rLm) vaccines expressing the Mycobacterium tuberculosis 30-kDa major secretory protein (r30/antigen 85B [Ag85B]) (rLm30) as heterologous booster vaccines in animals primed with BCG. Using three attenuated L. monocytogenes vectors, L. monocytogenes ΔactA (LmI), L. monocytogenes ΔactA ΔinlB (LmII), and L. monocytogenes ΔactA ΔinlB prfA* (LmIII), we constructed five rLm30 vaccine candidates expressing r30 linked in frame to the L. monocytogenes listeriolysin O signal sequence and driven by the hly promoter (h30) or linked in frame to the ActA N-terminal 100 amino acids and driven by the actA promoter (a30). All five rLm30 vaccines secreted r30 in broth and macrophages; while rLm30 expressing r30 via a constitutively active prfA* regulon (rLmIII/a30) expressed the largest amount of r30 in broth culture, all five rLm30 vaccines expressed equivalent amounts of r30 in infected macrophages. In comparative studies, boosting of BCG-immunized mice with rLmIII/a30 induced the strongest antigen-specific T-cell responses, including splenic and lung polyfunctional CD4+ T cells expressing the three cytokines interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-2 (IL-2) (P < 0.001) and splenic and lung CD8+ T cells expressing IFN-γ (P < 0.0001). In mice and guinea pigs, the rLmIII/a30 and rLmI/h30 vaccines were generally more potent booster vaccines than r30 with an adjuvant and a recombinant adenovirus vaccine expressing r30. In a setting in which BCG alone was highly immunoprotective, boosting of mice with rLmIII/a30, the most potent of the vaccines, significantly enhanced protection against aerosolized M. tuberculosis (P < 0.01).

scholarly journals rBCG30-Induced Immunity and Cross-Protection against Mycobacterium leprae Challenge Are Enhanced by Boosting with the Mycobacterium tuberculosis 30-Kilodalton Antigen 85B

2014 ◽  
Vol 82 (9) ◽  
pp. 3900-3909 ◽  
Author(s):  
Thomas P. Gillis ◽  
Michael V. Tullius ◽  
Marcus A. Horwitz
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Immune Responses ◽  
Mycobacterium Bovis ◽  
Protective Efficacy ◽  
Mycobacterium Leprae ◽  
Secretory Protein ◽  
Cross Protection ◽  
Content Type ◽  
Antigen 85B ◽  
The One

ABSTRACTLeprosy remains a major global health problem and typically occurs in regions in which tuberculosis is endemic. Vaccines are needed that protect against both infections and do so better than the suboptimalMycobacterium bovisBCG vaccine. Here, we evaluated rBCG30, a vaccine previously demonstrated to induce protection superior to that of BCG againstMycobacterium tuberculosisandMycobacterium bovischallenge in animal models, for efficacy againstMycobacterium lepraechallenge in a murine model of leprosy. rBCG30 overexpresses theM. tuberculosis30-kDa major secretory protein antigen 85B, which is 85% homologous with theM. lepraehomolog (r30ML). Mice were sham immunized or immunized intradermally with BCG or rBCG30 and challenged 2.5 months later by injection of viableM. lepraeinto each hind footpad. After 7 months, vaccine efficacy was assessed by enumerating theM. lepraebacteria per footpad. Both BCG and rBCG30 induced significant protection againstM. lepraechallenge. In the one experiment in which a comparison between BCG and rBCG30 was feasible, rBCG30 induced significantly greater protection than did BCG. Immunization of mice with purifiedM. tuberculosisorM. lepraeantigen 85B also induced protection againstM. lepraechallenge but less so than BCG or rBCG30. Notably, boosting rBCG30 withM. tuberculosisantigen 85B significantly enhanced r30ML-specific immune responses, substantially more so than boosting BCG, and significantly augmented protection againstM. lepraechallenge. Thus, rBCG30, a vaccine that induces improved protection againstM. tuberculosis, induces cross-protection againstM. lepraethat is comparable or potentially superior to that induced by BCG, and boosting rBCG30 with antigen 85B further enhances immune responses and protective efficacy.

scholarly journals Protein profile as a quality indicator of cryopreserved semen from Tambaqui Colossoma macropomum (Cuvier, 1818)

2020 ◽  
Vol 80 (4) ◽  
pp. 752-762
Author(s):  
J. M. Galo ◽  
D. P. Streit-Jr ◽  
C. D. Corcini ◽  
A. S. Varela-Jr ◽  
R. D. Jardim ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Sperm Quality ◽  
Protein Profile ◽  
Great Influence ◽  
Fertilization Rate ◽  
Colossoma Macropomum ◽  
One Dimensional ◽  
Qualitative And Quantitative ◽  
Sds Page ◽  
Dimensional Electrophoresis

Abstract The aim of this study was to evaluate the association between proteins in the seminal plasma of tambaqui Colossoma macropomum (Cuvier, 1818) with seminal quality indicators after thawing. The semen was cryopreserved with a dilution based on BTS with 8% DMSO. A 200 µL sample of semen from each animal was diluted in 800 µL BTS, centrifuged at 800 rpm, and the supernatant was cryopreserved to further analyze of the protein profile of seminal plasma through one-dimensional electrophoresis (SDS-PAGE). After 15 days of cryopreservation, a cryopreserved semen straw was thawed to analyze both qualitative and quantitative parameters. When considering all collections, the SDS-PAGE identified 15 protein bands in the seminal plasma of tambaqui. When the interaction (presence or absence) between proteins observed in the seminal plasma and the post thawed spermatic parameters was evaluated, we observed a great influence of the presence of proteins on spermatic quality. A greater (P<0.05) fertilization rate was observed with the presence of proteins 12, 34, 44, 85, and 90 kDa. Proteins in seminal plasma of tambaqui influenced the spermatic quality after thawing, and thus, they can be utilized as an indicator of sperm quality, especially the proteins with a molecular weight ≤ 50 kDa.

scholarly journals Protein Extraction Protocol from Musa sp. Shoots and Roots Tissue for Non-reducing One Dimensional SDS-PAGE Analysis

2018 ◽  
Vol 8 (3) ◽  
pp. 191-195
Author(s):  
Nurul N.M. Nasir ◽  
Ho Chai-Ling ◽  
Dhilia U. Lamasudin ◽  
Noor B. Saidi
Keyword(s):  
Protein Extraction ◽  
One Dimensional ◽  
Extraction Protocol ◽  
Musa Sp ◽  
Sds Page

scholarly journals Enhancing the Protective Efficacy of Mycobacterium bovis BCG Vaccination against Tuberculosis by Boosting with the Mycobacterium tuberculosis Major Secretory Protein

2005 ◽  
Vol 73 (8) ◽  
pp. 4676-4683 ◽  
Author(s):  
Marcus A. Horwitz ◽  
Günter Harth ◽  
Barbara Jane Dillon ◽  
Saša Masleša-Galić
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Pulmonary Tuberculosis ◽  
Guinea Pig ◽  
Mycobacterium Bovis ◽  
Protective Immunity ◽  
Protective Efficacy ◽  
Secretory Protein ◽  
Pig Model ◽  
Booster Vaccine ◽  
Guinea Pig Model

ABSTRACT Tuberculosis continues to ravage humanity, killing 2 million people yearly. Most cases occur in areas of the world to which the disease is endemic, where almost everyone is vaccinated early in life with Mycobacterium bovis BCG, the currently available vaccine against tuberculosis. Thus, while more-potent vaccines are needed to replace BCG, new vaccines are also needed to boost the immune protection of the 4 billion people already vaccinated with BCG. Until now, no booster vaccine has been shown capable of significantly enhancing the level of protective immunity induced by BCG in the stringent guinea pig model of pulmonary tuberculosis, the “gold standard” for testing tuberculosis vaccines. In this paper, we describe a booster vaccine for BCG comprising the purified recombinant Mycobacterium tuberculosis 30-kDa protein, the major secreted protein of this pathogen. In the guinea pig model of pulmonary tuberculosis, boosting BCG-immunized animals once with the 30-kDa protein greatly increased cell-mediated and humoral immune responses to the protein in three consecutive experiments. Most importantly, boosting BCG-immunized animals once with the 30-kDa protein significantly enhanced protective immunity against aerosol challenge with highly virulent M. tuberculosis, as evidenced by a significantly reduced lung and spleen burden of M. tuberculosis compared with those for nonboosted BCG-immunized animals (mean additional reduction in CFU of 0.4 ± 0.1 log in the lung [P = 0.03] and 0.6 ± 0.1 log in the spleen [P = 0.002]). This study suggests that administering BCG-immunized people a booster vaccine comprising the 30-kDa protein may enhance their level of immunoprotection against tuberculosis.

scholarly journals Protection Elicited by Two Glutamine Auxotrophs of Mycobacterium tuberculosis and In Vivo Growth Phenotypes of the Four Unique Glutamine Synthetase Mutants in a Murine Model

2006 ◽  
Vol 74 (11) ◽  
pp. 6491-6495 ◽  
Author(s):  
Sunhee Lee ◽  
Bo-Young Jeon ◽  
Svetoslav Bardarov ◽  
Mei Chen ◽  
Sheldon L. Morris ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Glutamine Synthetase ◽  
Mycobacterium Bovis ◽  
Murine Model ◽  
Triple Mutant ◽  
Auxotrophic Mutants ◽  
Bcg Vaccination ◽  
In Vivo Growth ◽  
Subcutaneous Immunization

ABSTRACT We generated four individual glutamine synthetase (GS) mutants (ΔglnA1, ΔglnA2, ΔglnA3, and ΔglnA4) and one triple mutant (ΔglnA1EA2) of Mycobacterium tuberculosis to investigate the roles of GS enzymes. Subcutaneous immunization with the ΔglnA1EA2 and ΔglnA1 glutamine auxotrophic mutants conferred protection on C57BL/6 mice against an aerosol challenge with virulent M. tuberculosis, which was comparable to that provided by Mycobacterium bovis BCG vaccination.

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