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1.
J Bacteriol ; 172(2): 519-24, 1990 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-2153655

RESUMO

A system that permits molecular genetic manipulation of mycobacteria was developed on the basis of the yeast paradigm of gene replacement by homologous recombination. A shuttle vector that can replicate autonomously at a high copy number in Escherichia coli but must integrate into homologous DNA for survival in Mycobacterium smegmatis was constructed. The vector contains a ColE1 origin of replication, antibiotic resistance markers for ampicillin and kanamycin, a nutritional marker (pyrF) that allows both positive and negative selection in E. coli and M. smegmatis, and unique restriction sites that permit insertion of foreign DNA. Transformation of mycobacteria with this vector results in integration of its DNA into the genomic pyrF locus by either a single or a double homologous recombination event. With this system, the 65-kilodalton Mycobacterium leprae stress protein antigen was inserted into the M. smegmatis genome and expressed. This gene replacement technology, together with a uniquely useful pyrF marker, should be valuable for investigating mycobacterial pathobiology, for the development of candidate mycobacterial vaccine vehicles, and as a model for the development of molecular genetic systems in other pathogenic microorganisms.


Assuntos
DNA Bacteriano/genética , Escherichia coli/genética , Engenharia Genética/métodos , Mycobacterium/genética , Southern Blotting , Genes Bacterianos , Vetores Genéticos , Biblioteca Genômica , Proteínas de Choque Térmico/genética , Canamicina Quinase , Mycobacterium leprae/genética , Fosfotransferases/genética , Transformação Bacteriana
2.
J Bacteriol ; 170(12): 5919-21, 1988 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-3056923

RESUMO

The gene encoding an immunologically important 18-kilodalton protein antigen of Mycobacterium leprae has been sequenced, and the amino acid sequence of the antigen has been deduced. The 18-kilodalton antigen is strikingly similar in size and sequence to a family of eucaryotic heat shock proteins.


Assuntos
Antígenos de Bactérias/genética , Genes Bacterianos , Genes , Proteínas de Choque Térmico/genética , Mycobacterium leprae/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Bacteriano/genética , Dados de Sequência Molecular , Mycobacterium leprae/imunologia , Plantas/genética , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Soja
3.
J Immunol ; 141(12): 4370-5, 1988 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-3058804

RESUMO

The gene for a 28-kDa Mycobacterium leprae protein Ag, a major target of antibodies from patients with lepromatous leprosy, was cloned from a lambda-gt11-M. leprae DNA expression library and sequenced. Antibodies to this protein were detected in the serum of the majority of 15 individual lepromatous patients that were tested. The predicted amino acid sequence of the 28-kDa protein suggests that it is localized to the bacterial plasma membrane or cell wall.


Assuntos
Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/imunologia , Hanseníase Virchowiana/imunologia , Mycobacterium leprae/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Sequência de Bases , Clonagem Molecular , Genes Bacterianos , Humanos , Sondas Moleculares , Dados de Sequência Molecular , Peso Molecular , Mycobacterium leprae/genética , Proteínas Recombinantes de Fusão/imunologia
4.
J Immunol ; 141(8): 2729-33, 1988 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-2459226

RESUMO

We have used human CD4+ T lymphocyte clones as primary probes to identify and isolate lambda gt11 rDNA clones that express epitopes recognized by T cells. The method that we describe here permits a direct survey of T cell epitope coding sequences in genomic DNA or cDNA libraries. A lambda gt11 library of Mycobacterium leprae DNA was screened with M. leprae-reactive human T cell clones as probes, allowing the isolation of a M. leprae DNA clone encoding the unidentified Ag. This DNA clone differs in restriction maps from those previously identified by antibody probes and encodes an epitope that is unique to vaccine strains of Mycobacterium bovis bacillus Calmette-Guérin and pathogenic mycobacteria. This method is generally applicable and should expedite the study of Ag and epitopes important to the T cell response in infections and in autoimmune diseases.


Assuntos
Antígenos de Bactérias/genética , Sondas de DNA , Genes Bacterianos , Infecções por Mycobacterium/genética , Mycobacterium bovis/genética , Linfócitos T/análise , Antígenos de Bactérias/imunologia , Antígenos de Diferenciação de Linfócitos T/genética , Clonagem Molecular/métodos , DNA/imunologia , DNA Recombinante , Epitopos/genética , Epitopos/imunologia , Humanos , Ativação Linfocitária , Infecções por Mycobacterium/imunologia , Mycobacterium bovis/imunologia , Linfócitos T/imunologia
5.
Proc Natl Acad Sci U S A ; 85(12): 4267-70, 1988 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-3132709

RESUMO

To understand the immune response to infection by tuberculosis and leprosy bacilli and to develop improved vaccines, the nature of antigens that are involved in humoral and cell-mediated immunity was investigated. We have determined that five immunodominant protein antigens under study are homologues of stress proteins. This finding and observations with other pathogens suggest that infectious agents may respond to the host environment by producing stress proteins and that these proteins can be important immune targets. We postulate that abundant and highly conserved stress proteins may have "immunoprophylactic" potential for a broad spectrum of human pathogens.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Choque Térmico/imunologia , Hanseníase/imunologia , Mycobacterium leprae/genética , Mycobacterium tuberculosis/genética , Tuberculose/imunologia , Sequência de Aminoácidos , Clonagem Molecular , Reações Cruzadas , Escherichia coli/genética , Genes , Genes Bacterianos , Proteínas de Choque Térmico/genética , Humanos , Dados de Sequência Molecular , Mycobacterium leprae/imunologia , Mycobacterium tuberculosis/imunologia
7.
EMBO J ; 6(5): 1245-9, 1987 May.
Artigo em Inglês | MEDLINE | ID: mdl-2440673

RESUMO

Two complementary approaches were used to determine the epitope specificity of clonal and polyclonal human T lymphocytes reactive with the 65-kd antigen of Mycobacterium leprae. A recombinant DNA sublibrary constructed from portions of the 65-kd gene was used to map T cell determinants within amino acid sequences 101-146 and 409-526. Independently, potential T cell epitopes within the protein were predicted based on an empirical analysis of specific patterns in the amino acid sequence. Of six peptides that were predicted and subsequently synthesised, two (112-132 and 437-459) were shown to contain human T cell epitopes. This corroborated and refined the results obtained using the recombinant DNA sublibrary. Both of these regions are identical in M. leprae and M. tuberculosis and are distinct from the known B cell epitopes of the 65-kd protein. This combination of recombinant DNA technology and peptide chemistry may prove valuable in analysis of the cellular immune response to infectious agents.


Assuntos
Antígenos de Bactérias/genética , Epitopos/análise , Mycobacterium leprae/imunologia , Mycobacterium tuberculosis/genética , Proteínas Recombinantes/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Antígenos de Bactérias/imunologia , Clonagem Molecular , Colífagos/genética , DNA Recombinante/metabolismo , Escherichia coli/genética , Genes , Humanos , Mycobacterium leprae/genética , Mycobacterium tuberculosis/imunologia
8.
Infect Immun ; 55(4): 1000-3, 1987 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-2435658

RESUMO

Twenty-three monoclonal antibodies (MAbs) prepared in seven different laboratories were studied, all of which recognized the 65-kilodalton (kDa) protein of Mycobacterium leprae as determined by Western blotting or gel radioimmunoassay or both. Fourteen of the MAbs recognized different epitopes, as evaluated by cross-competition studies using radiolabeled MAb and unlabeled inhibitors; the species specificity of these epitopes was defined by nitrocellulose dot blot immunoassays with bacterial sonic extract antigen preparations from 23 species of mycobacteria. Each of the 14 distinct MAbs recognized a 65-kDa protein produced by a lysogenized Escherichia coli Y1089 host containing cloned rDNA which included the gene for the M. leprae 65-kDa protein. Of the 14 distinct MAbs, 1 recognized an epitope found only on M. leprae, and the others recognized epitopes present on as few as 8 or as many as all 23 of the mycobacterial species studied. Identification of these distinct 65-kDa protein epitopes and use of the MAbs which recognize them should assist future structural studies of this protein and characterization of the T-cell reactive and serodiagnostically useful portions of the molecule.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Mycobacterium leprae/imunologia , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Ligação Competitiva , Epitopos , Peso Molecular
9.
Proc Natl Acad Sci U S A ; 84(6): 1679-83, 1987 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-3104901

RESUMO

Mycobacterium tuberculosis genes encoding immunologically relevant proteins were isolated by systematically screening a lambda gt11 recombinant DNA expression library with a collection of murine monoclonal antibodies directed against protein antigens of this pathogen. These antibodies, previously characterized by a World Health Organization workshop on monoclonal antibodies against mycobacteria, were used to isolate DNA sequences encoding five major protein antigens of this pathogen. To evaluate the extent of crossreactivity between these M. tuberculosis antigens and antigens of Mycobacterium leprae, recombinant antigens were probed with monoclonal antibodies directed against the protein antigens of these bacilli. One of the antigens, a 65-kDa protein, has determinants common to M. tuberculosis and M. leprae. We find not only that this antigen is recognized by mouse monoclonal antibodies but that it is the major protein recognized by anti-M. tuberculosis rabbit sera. The 65-kDa proteins of M. tuberculosis and M. leprae appear to play a role in the humoral and cell-mediated immune response to these pathogens.


Assuntos
Antígenos de Bactérias/genética , Genes Bacterianos , Mycobacterium leprae/imunologia , Mycobacterium tuberculosis/imunologia , Animais , Reações Cruzadas , Mycobacterium leprae/genética , Mycobacterium tuberculosis/genética , Coelhos , Proteínas Recombinantes/imunologia
14.
Proc Natl Acad Sci U S A ; 83(18): 7013-7, 1986 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2428046

RESUMO

A recombinant DNA expression strategy has been used to deduce the amino acid sequences of six different antigenic determinants in a single protein of Mycobacterium leprae, the etiologic agent of leprosy. The gene encoding the M. leprae 65-kDa antigen was sequenced and a lambda gt11 gene sublibrary was constructed with fragments of the gene. Recombinant DNA clones producing specific antigenic determinants were isolated by screening with monoclonal antibodies, and the sequences of their insert DNAs were determined with a rapid primer-extension method. The amino acid sequence of each determinant was deduced from the minimum overlap of insert DNAs from multiple antibody-positive DNA clones. Amino acid sequences for six different epitopes were elucidated. A peptide containing sequences for one of these epitopes was synthesized and shown to bind the appropriate monoclonal antibody; this antigenic determinant is unique to M. leprae. The approach described here can be used to rapidly elucidate protein epitopes that are recognized by antibodies or T cells.


Assuntos
Antígenos de Bactérias/análise , Epitopos/análise , Mycobacterium leprae/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/genética , Sequência de Bases , DNA Bacteriano/análise , Humanos , Hanseníase/imunologia
15.
Nature ; 319(6048): 63-6, 1986.
Artigo em Inglês | MEDLINE | ID: mdl-3510397

RESUMO

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. As with other intracellular parasites, protective immunity is dependent on T cells and cell-mediated immunity. In animal models, immunization with killed armadillo-derived M. leprae elicits strong T-cell responses, delayed-type hypersensitivity and protection against viable challenge. We have recently shown that killed M. leprae can induce delayed-type hypersensitivity in healthy human volunteers. Identification of the M. leprae antigens that are recognized by T cells and may be involved in protection has been hampered by the inability to cultivate the organism in vitro and by difficulties in antigen purification from limited quantities of armadillo-derived bacillus. Because genes for the major protein antigens of M. leprae as seen by mouse monoclonal antibodies have been isolated, it has become possible to test whether these individual antigens are recognized by T cells. We screened crude lambda gtll phage lysates of Escherichia coli containing individual M. leprae antigens using M. leprae-specific T-cell clones isolated from M. leprae-vaccinated volunteers. Using this method, we find that nearly half of the M. leprae-specific T-cell clones are stimulated to proliferate by lysates containing an epitope of a M. leprae protein of relative molecular mass 18,000 (18K).


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Mycobacterium leprae/imunologia , Proteínas Recombinantes/imunologia , Linfócitos T/imunologia , Anticorpos Monoclonais , Células Clonais/imunologia , Escherichia coli , Humanos , Ativação Linfocitária , Peso Molecular
16.
Biosci Rep ; 5(10-11): 839-45, 1985.
Artigo em Inglês | MEDLINE | ID: mdl-3006821

RESUMO

The lambda gt 11 expression vector permitted us to survey protein antigens of Mycobacterium leprae and Mycobacterium tuberculosis expressed in Escherichia coli. Using monoclonal antibodies, recombinant clones were detected producing three major antigens of M. tuberculosis and five major protein antigens of M. leprae. These recombinant antigens produced in E. coli should prove useful for diagnosis, epidemiology and possibly the development of recombinant mycobacterial vaccines.


Assuntos
Antígenos Virais/genética , Genes Bacterianos , Genes , Mycobacterium leprae/genética , Mycobacterium tuberculosis/genética , Clonagem Molecular , Enzimas de Restrição do DNA , DNA Recombinante/metabolismo , Escherichia coli/genética , Vetores Genéticos
17.
Nature ; 316(6027): 450-2, 1985.
Artigo em Inglês | MEDLINE | ID: mdl-3894979

RESUMO

Leprosy, a chronic infectious disease afflicting between 10 and 15 million people, is caused by the obligate intracellular parasite Mycobacterium leprae. Although M. leprae was the first identified bacterial pathogen of man, basic biochemical, immunological, diagnostic and therapeutic investigations have been severely limited because it remains one of the few human pathogens that have not been cultured in vitro. An M. leprae recombinant DNA expression library was constructed to provide a source of genes encoding proteins relevant for such studies. Monoclonal antibodies directed against M. leprae specific antigens have been used to isolate the genes encoding the five most immunogenic protein antigens of the leprosy bacillus. We report here that M. leprae specific epitopes recognized by all of 13 monoclonal antibodies tested were produced by recombinant phage in Escherichia coli.


Assuntos
Antígenos de Bactérias/genética , Genes Bacterianos , Genes , Mycobacterium leprae/genética , Anticorpos Monoclonais , Sequência de Bases , Clonagem Molecular , DNA Recombinante/análise , Humanos , Imunidade Celular , Hanseníase/imunologia , Mycobacterium leprae/imunologia
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