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1.
Saudi Medical Journal. 2014; 35 (4): 350-359
in English | IMEMR | ID: emr-159351

ABSTRACT

To clone and express Mycobacterium tuberculosis [M. tuberculosis] proteins PE35 and culture filtrate protein [CFP]10 in Mycobacterium vaccae [M. vaccae], and subsequently, evaluate the humoral and cellular immunity responses against these recombinant constructs in mice. The DNA of PE35 and CFP 10 genes were cloned into the shuttle plasmid pDE22, and the recombinant plasmids were electroporated into M. vaccae. The recombinant constructs were then tested for expression of PE35 and CFP10 by Western immunoblotting using rabbit anti-sera. Furthermore, splenocytes and sera from groups of 5 mice immunized with recombinant M. vaccae [rVaccae] were tested for cellular and humoral responses in proliferation, and antibody assays. Experiments were carried out in the laboratory of the Faculty of Medicine, Kuwait University, Safat, Kuwait between 2009 and 2011. The results of Western immunoblot suggested the expression of only PE35. However, splenocyte assays showed positive proliferation in response to peptide pools, and 4 and 5 of the 6 overlapping synthetic peptides covering the sequence of PE35 and CFP10. In addition, positive antibody reactivity was detected with PE35 peptide pool and a single peptide, namely, P2. The expression of PE35 and CFP10 proteins in rVaccae constructs led to the induction of cellular immune responses to multiple epitopes

2.
Medical Principles and Practice. 2008; 17 (4): 325-330
in English | IMEMR | ID: emr-88995

ABSTRACT

To evaluate cell-mediated immune [CMI] response in diabetic and non-diabetic tuberculosis [TB] patients and healthy subjects in response to complex, fractionated and single antigens of Mycobacterium tuberculosis. Peripheral blood mononuclear cells [PBMC] were obtained from patients suffering from pulmonary TB and type II diabetes [n = 7], pulmonary TB without diabetes [n = 10] and healthy subjects without TB and diabetes [n = 10]. PBMC were assessed for CMI responses in antigen-induced proliferation assays in response to complex mycobacterial antigens [whole cells, cell walls and culture filtrate of M. tuberculosis], a battery of naturally purified or recombinant produced secreted [ESAT6, MPT59, MPT64 and MTB38] and cytosolic [MTB10, MTB70, ML10, ML28, ML36, ML65 and MB65] mycobacterial antigens and fractionated culture filtrate proteins [fractions F1-F10] of M. tuberculosis. The majority [>70%] of diabetic and non-diabetic TB patients and healthy subjects responded to the complex antigens of M. tuberculosis. However, among the single antigens, ESAT6 was most frequently recognized by TB patients with and without diabetes, but least recognized by healthy subjects. The secreted antigens MPT59 and MPT64 were recognized by all the groups, whereas the cytosolic antigens were recognized best by healthy subjects. When tested with fractionated secreted proteins present in the culture filtrate of M. tuberculosis, the best responses in both diabetic and non-diabetic TB patients were obtained with fractions containing low-molecular-weight proteins. Diabetic and non-diabetic TB patients respond frequently to secreted low-molecular-weight ESAT6 antigen of M. tuberculosis, indicating that this antigen may be useful in the diagnosis of TB in both the groups


Subject(s)
Humans , Diabetes Mellitus , Mycobacterium tuberculosis/immunology , Antigens, Bacterial , Immunity, Cellular , Tuberculosis/immunology , Leukocytes, Mononuclear , Bacterial Proteins
3.
Medical Principles and Practice. 2008; 17 (5): 378-384
in English | IMEMR | ID: emr-89005

ABSTRACT

To amplify, clone and express in Escherichia coli six open reading frames [ORFs] predicted in the RD1 DNA segment of Mycobacterium tuberculosis and purify the expressed proteins to homogeneity. DNA corresponding to the coding regions of six RD1 ORFs, i.e. ORF10 to ORF15, was amplified from genomic DNA of M. tuberculosis, cloned in the plasmid vector pPCR-Script and subcloned in expression plasmid vectors pET29a and/or pGEX-4T for expression in E. coli as fusion proteins. The recombinant fusion proteins were identified by sodium dodecyl polyacrylamide gel electrophoresis and Western immunoblotting. Attempts were made to obtain purified proteins, free of the fusion partner, using affinity and fast protein liquid chromatography. DNA corresponding to all six targeted RD1 ORFs was amplified from the genomic DNA of M. tuberculosis and five of the six ORFs, with the exception of ORF13, were cloned in the plasmid vectors and expressed in E. coli. Because of extensive degradation of ORF10 and ORF12 fusion proteins or nonbinding to the affinity columns of ORF15 fusion proteins, only ORF11 and ORF14 proteins were purified, free of the fusion partner, to homogeneity. All of the six targeted RD1 genes were amplified and five expressed using E. coli hosts, but only two of the expressed proteins were purified to homogeneity. Alternative expression systems are required to obtain all RD1 proteins for functional characterization


Subject(s)
Gene Amplification , Genetic Vectors , Cloning, Molecular , Escherichia coli , Proteins/isolation & purification , DNA , Plasmids , Recombinant Fusion Proteins , Electrophoresis, Polyacrylamide Gel , Blotting, Western , Chromatography, Liquid
4.
Medical Principles and Practice. 2006; 15 (2): 137-144
in English | IMEMR | ID: emr-79527

ABSTRACT

To identify transcriptionally active open reading frames [ORFs], predicted by bioinformatics, within RD1 genomic segment of Mycobacterium tuberculosis using reverse transcription-polymerase chain reaction [RT-PCR]. M. tuberculosis H37Rv was grown in Middlebrook 7H9 medium for 8 weeks and total RNA was isolated using standard procedures. The cDNA was synthesized using first-strand cDNA synthesis kit and general primers provided in the kit [pd [N][6], and/or Not I-d[T][18]] as well as forward primers specific for each predicted RD1 ORF. Specific forward and reverse primers in PCR were used to amplify ORF-specific cDNA. The amplified products were identified on the basis of size using agarose gel electrophoresis, and their identity was confirmed by DNA sequencing. RT-PCR demonstrated expression of 13 of the 14 bioinformatics-predicted ORFs within RD1 genomic segment of M. tuberculosis. However, cDNA synthesis and PCR amplifications of specific products varied with respect to primer requirement and reaction conditions, respectively. All ORFs of <1.5 kb were amplified in standard RT-PCR, whereas several large-size ORFs [>1.5 kb] required internal primers for amplification in semi-nested RT-PCR. The sequencing of RT-PCR-amplified products of ORFs confirmed their identity. Bioinformatics analysis of DNA can accurately predict ORFs within M. tuberculosis-specific genomic regions, and RT-PCR is a suitable technique to confirm their expression in bacteria


Subject(s)
Humans , Bacterial Proteins/genetics , Open Reading Frames , DNA, Bacterial , Transcription, Genetic , Genes, Bacterial
5.
Medical Principles and Practice. 2005; 14 (3): 140-6
in English | IMEMR | ID: emr-73518

ABSTRACT

To identify T-cell epitopes of Ag85B by analysis of its sequence for prediction to bind HLA-DR alleles and evaluate the predicted peptides for recognition by T cells in antigen-induced proliferation assays. Materials/Subjects and The complete sequence of Ag85B was analyzed for HLA-DR binding prediction to 51 HLA-DR alleles by using a virtual matrix-based prediction program [ProPred]. Synthetic peptides covering the sequence of mature Ag85B were also analyzed for binding to HLA-DR alleles, and evaluated for recognition in antigen-induced proliferation assays with Ag85B-specific T-cell lines established from the peripheral blood mononuclear cells of 10 HLA-DR-heterogeneous tuberculosis patients. The ProPred analysis of the full-length Ag85B [325 aa], signal peptide [40 aa] and the mature protein [285 aa] predicted their binding to 100, 76 and 98% of the 51 HLA-DR alleles, respectively. The analysis of 31 synthetic peptides for binding to HLA-DR alleles showed that 4 of them could bind >50% HLA-DR alleles, and were considered promiscuous. Testing of Ag85B-specific T-cell lines with synthetic peptides showed that all of the T-cell lines responded to one or more peptides of Ag85B, and 9 of the 10 cell lines responded to one or more of the four peptides considered promiscuous for binding to HLA-DR alleles. The ProPred program was useful in predicting the HLA-DR alleles binding regions of Ag85B and identifying the promiscuous peptides recognized by T cells


Subject(s)
Humans , Antigens, Bacterial/metabolism , HLA-DR Antigens , Epitopes, T-Lymphocyte , Bacterial Proteins/metabolism , T-Lymphocytes/immunology
6.
Medical Principles and Practice. 1997; 6 (2): 103-109
in English | IMEMR | ID: emr-45957

ABSTRACT

In recent years, several defined antigens of Mycobacterium tuberculosis have become available either by purifying the natural antigens using biochemical procedures, or by producing large quantities of recombinant antigens, using recombinant DNA technology. In this study, we evaluated these defined antigens of M. tuberculosis, along with the complex antigens, for humoral immune responses in an IgG-specific enzyme-linked immunosorbent assay [ELISA]. We hoped to develop a tuberculosis-specific ELISA of diagnostic potential. Our results show that IgG antibodies to the complex antigens [i.e. culture filtrate, cell wall and sonicate] and to some of the defined antigens [i.e. 38-kD antigen and lipoarabinomannan] of M. tuberculosis were significantly elevated in the sera of tuberculous patients, as compared to healthy contacts of tuberculous patients and patients suffering from nonmycobacterial infections. Among the IgG subclasses, IgG2 antibody reactive with culture filtrate, cell wall, and lipoarabinomannan antigens was elevated in the sera of most tuberculous patients. Since the complex antigens are difficult to standardize, and bacth-to-batch variations may occur with respect to the amount of active components in different batches, the use of defined antigens of M. tuberculosis in ELISA may be a better alternative in the early and specific diagnosis of tuberculosis


Subject(s)
Humans , Antigens , Mycobacterium tuberculosis/immunology , Enzyme-Linked Immunosorbent Assay , Immunoglobulin G/blood , Evaluation Study/methods , Regression Analysis
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