[Construction of recombinant Mycobacterium smegmatis expressing ESAT-6 and its effects on macrophages].
OBJECTIVE
Objective To construct recombinant Mycobacterium smegmatis expressing ESAT-6 of the human pathogen Mycobacterium tuberculosis.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Li
Yan
Y
Laboratory of Infection and Immunity, College of Preclinical and Forensic Medicine, West China Medical Center of Sichuan University, Chengdu 610041, China. ly506@163.com
Bao
Lang
L
Zhang
Hui-dong
HD
Li
Ya-sha
YS
Zhu
Hai-long
HL
D000818:Animals; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D017209:Apoptosis / Q000276:immunology; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D008262:Macrophage Activation / Q000276:immunology; D008264:Macrophages / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D051379:Mice; D020102:Mycobacterium smegmatis / Q000235:genetics* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014170:Transformation, Genetic
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D012333:RNA, Messenger; D011994:Recombinant Proteins; D052247:Nitric Oxide Synthase Type II