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1.
Vaccine ; 20(5-6): 731-6, 2001 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-11738736

RESUMO

Expression vectors containing rabies virus nucleoprotein B-cell and T-cell epitopes in Mycobacterium bovis BCG were constructed. The epitopes were subcloned into the M. leprae 18-kDa gene to ensure correct presentation to the host immune system. Expression of the 18-kDa::B+T epitope fusion protein was driven by either the hsp60 promoter, which is constitutively activated at a high level in M. bovis BCG, or the 18-kDa promoter, which is strongly induced in vivo. Mice were immunised intra-peritoneally with the recombinant BCG cultures and compared to a control group vaccinated with the commercial rabies vaccine Rai-SAD. Both of the expression vectors elicited a higher antibody titre than that of the rabies vaccine, with the highest response shown by M. bovis BCG (pUP203), expression controlled by the 18-kDa promoter. Immunisation with M. bovis BCG (pUP202), expression controlled by the hsp60 promoter, resulted in a continuously increasing antibody titre up to 60 days post immunisation. The mice antibodies were also capable of recognising the whole rabies virus and not only the synthetic peptide epitopes.


Assuntos
Antígenos Virais/genética , Mycobacterium bovis/genética , Mycobacterium bovis/imunologia , Nucleocapsídeo/genética , Nucleocapsídeo/imunologia , Vírus da Raiva/genética , Vírus da Raiva/imunologia , Animais , Anticorpos Antivirais/biossíntese , Linfócitos B/imunologia , Sequência de Bases , Epitopos/genética , Expressão Gênica , Vetores Genéticos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Nucleocapsídeo , Plasmídeos/genética , Vacinas Antirrábicas/genética , Vacinas Antirrábicas/imunologia , Linfócitos T/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
2.
J Bacteriol ; 176(9): 2525-31, 1994 May.
Artigo em Inglês | MEDLINE | ID: mdl-7909542

RESUMO

The causative agents of leprosy and tuberculosis, Mycobacterium leprae and Mycobacterium tuberculosis, have a lipid-rich cell envelope which contributes to virulence and antibiotic resistance. Acyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis, consists in mycobacteria of two subunits, one of which is biotinylated. Genes from M. leprae and M. tuberculosis encoding a biotinylated protein have been cloned and sequenced. Analysis of the derived protein sequences demonstrated the presence of biotin-binding sites and putative ATP-bicarbonate interactions sites, consistent with the proteins having a biotin carboxylase function as well as their being biotin carrier proteins.


Assuntos
Acetil-CoA Carboxilase/genética , Proteínas de Transporte/genética , Genes Bacterianos/genética , Lipídeos/biossíntese , Mycobacterium leprae/genética , Mycobacterium tuberculosis/genética , Sequência de Aminoácidos , Sequência de Bases , Biotina/análise , Sondas de DNA , Ácido Graxo Sintase Tipo II , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
3.
Mol Microbiol ; 10(5): 983-93, 1993 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-7934874

RESUMO

We have constructed a mycobacterial integrative vector by placing two copies of the insertion sequence IS900 flanking a kanamycin-resistance gene into a 'suicide' vector unable to replicate in mycobacteria. The Mycobacterium leprae gene encoding the M. leprae 18 kDa protein was cloned between the two copies of IS900 to provide expression signals. Constructs were introduced into Mycobacterium species smegmatis, vaccae and bovis BCG by electroporation and selection for kanamycin resistance. The expression of the 18 kDa gene was analysed by Western blotting. Integration of the vector into the M. smegmatis chromosome was analysed by Southern blotting. One to five copies of the vector were detected in each transformant. The SIV gag p27 gene and the foot-and-mouth disease virus VP1 140-160 epitope were successfully cloned into the 18 kDa gene and expression in M. smegmatis was obtained.


Assuntos
Elementos de DNA Transponíveis , Vetores Genéticos , Mycobacterium/genética , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Aphthovirus/genética , Sequência de Bases , Capsídeo/genética , Proteínas do Capsídeo , Clonagem Molecular , Primers do DNA/genética , DNA Viral/genética , Expressão Gênica , Produtos do Gene gag/genética , Genes Virais , Genes gag , Resistência a Canamicina/genética , Dados de Sequência Molecular , Mycobacterium leprae/genética , Mycobacterium leprae/imunologia , Vírus da Imunodeficiência Símia/genética , Transformação Genética
4.
Acta Leprol ; 7 Suppl 1: 212-6, 1989.
Artigo em Inglês | MEDLINE | ID: mdl-2503993

RESUMO

One limiting factor in the studies of tuberculosis and leprosy is the lack of a versatile system for genetic analysis and manipulation of mycobacteria. One strategy used in constructing a plasmid vector for transforming Mycobacterium smegmatis was to insert fragments of a mycobacterial plasmid into an Escherichia coli plasmid. We found that the parental E. coli plasmid is capable of self-replication in M. smegmatis yielding chloramphenicol-resistant colonies. Plasmids from different passages of one M. smegmatis transformant were recovered and characterised by restriction digest analysis. The plasmid from the earlier passage was found to be indistinguishable from the original plasmid by restriction analysis. Plasmids from later preparations, however, were found to have undergone modifications in the M. smegmatis host resulting in an apparent increase in transformation efficiency for M. smegmatis. These plasmids can be used as a shuttle vector for the genetic manipulation of mycobacterial species.


Assuntos
Mycobacterium/genética , Transformação Genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Escherichia coli/genética , Vetores Genéticos , Plasmídeos , Mapeamento por Restrição
5.
Mol Microbiol ; 3(1): 29-34, 1989 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-2654539

RESUMO

One limiting factor in studies of tuberculosis and leprosy is the difficulty of genetic analysis and manipulation of mycobacteria. Two approaches were adopted for the construction of vectors, based on different Escherichia coli plasmids and using Mycobacterium smegmatis as the host. In both cases we found that the original E. coli plasmid is capable of being replicated in M. smegmatis, yielding chloramphenicol-resistant colonies. One such plasmid has been recovered from a M. smegmatis transformant and used to re-transform both M. smegmatis and E. coli to chloramphenicol resistance. This plasmid is indistinguishable from the original plasmid by restriction analysis, and can be used as a shuttle vector for the genetic manipulation of mycobacterial species.


Assuntos
Resistência ao Cloranfenicol/genética , Escherichia coli/genética , Mycobacterium/genética , Plasmídeos , Transformação Genética , Southern Blotting , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Vetores Genéticos , Mapeamento por Restrição
6.
J Bacteriol ; 168(1): 72-80, 1986 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-3020007

RESUMO

The ability of "Streptomyces lividans" to use the expression signals of genes from Mycobacterium bovis BCG was tested in vivo by using gene fusions. Random DNA fragments from M. bovis BCG were inserted into promoter-probe plasmids in Escherichia coli and in "S. lividans." Comparison with promoter activity detected with random DNA fragments from the respective hosts suggested that "S. lividans" efficiently utilizes a high proportion of mycobacterial promoters, whereas a smaller fraction are expressed, and expressed more weakly, in E. coli. M. bovis BCG DNA fragments were also inserted into the specially constructed translational fusion vector (pIJ688) in "S. lividans." pIJ688 contains the kanamycin phosphotransferase gene (neo) from transposon Tn5, truncated at its amino terminus, as the indicator. The results suggested that "S. lividans" uses M. bovis BCG translational signals almost as efficiently as its own signals. Moreover, several hybrid proteins with an M. bovis BCG-derived amino terminus seemed to be reasonably stable in "S. lividans." These experiments indicate that "S. lividans" may be a suitable host for the expression of Mycobacterium leprae and Mycobacterium tuberculosis genes from their own signals. This is a precondition for the expression of entire biosynthetic pathways, which could be valuable in the production of diagnostic and therapeutic agents. The vectors may also have wider applications for the analysis of gene expression in Streptomyces.


Assuntos
Clonagem Molecular , Genes Bacterianos , Mycobacterium bovis/genética , Regiões Promotoras Genéticas , Streptomyces/genética , Elementos de DNA Transponíveis , Resistência Microbiana a Medicamentos , Escherichia coli/genética , Canamicina/farmacologia , Canamicina Quinase , Fosfotransferases/biossíntese , Fosfotransferases/genética , Biossíntese de Proteínas , Streptomyces/efeitos dos fármacos , Streptomyces/enzimologia
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