Construction of recombinant Mycobacterium smegmatis expressing ESAT-6 and its effects on macrophages / 南方医科大学学报
Journal of Southern Medical University
;
(12): 923-926, 2006.
Article
Dans Chinois
| WPRIM
| ID: wpr-282884
ABSTRACT
<p><b>OBJECTIVE</b>Objective To construct recombinant Mycobacterium smegmatis expressing ESAT-6 of the human pathogen Mycobacterium tuberculosis.</p><p><b>METHODS</b>ESAT-6 gene was amplified from M. tuberculosis genomic DNA and inserted into an E.coli-mycobacterium shuttle vector under the control of HSP60 promoter. The recombinant vector was transformed into M. smegmatis by electroporation. To assess the ability of recombinant M. smegmatis to activate macrophage, mouse macrophage ANA-1 was cocultured with recombinant M. smegmatis. The apoptosis of ANA-1 cells was detected by flow cytometry and iNOS mRNA expression of the cells was detected by reverse transcription-polymerase chain reaction (RT-PCR). The survival of M. smegmatis strains in ANA-1 cells was evaluated.</p><p><b>RESULTS</b>The recombinant vector was verified by restriction endonuclease digestion and DNA sequencing. ESAT-6 protein was expressed in M. smegmatis in response to heat shock and the molecular weight of the expression product was identical to the expected value. The growth curve of the new recombinant M. smegmatis was consistent with that of the wild-type strain, suggesting the absence of ESAT-6 protein toxicity against M. smegmatis. The recombinant M. smegmatis did not induce significant changes in mouse macrophage ANA-1 apoptosis. Coculture of the macrophages with recombinant M. smegmatis for 4 to 24 h could induce iNOS expression in the former, and the CFU of recombination M. smegmatis grown in ANA-1 cells was much less than that of the control bacteria.</p><p><b>CONCLUSION</b>The recombinant M. smegmatis expressing M. tuberculosis ESAT-6 gene possess immunogenicity, which provides experimental evidence for the development of novel M. smegmatis-based vaccine against tuberculosis.</p>
Texte intégral:
Disponible
Indice:
WPRIM (Pacifique occidental)
Sujet Principal:
Protéines bactériennes
/
Transformation génétique
/
Protéines recombinantes
/
ARN messager
/
Lignée cellulaire
/
Apoptose
/
Mycobacterium smegmatis
/
RT-PCR
/
Biologie cellulaire
/
Électrophorèse sur gel de polyacrylamide
Limites du sujet:
Animaux
/
Humains
langue:
Chinois
Texte intégral:
Journal of Southern Medical University
Année:
2006
Type:
Article
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