Attenuated Salmonella enterica serovar typhi live vector with inducible chromosomal expression of the T7 RNA polymerase and its evaluation with reporter genes.
Attenuated Salmonella strains with defined gene deletions have been extensively evaluated as suitable live carriers of passenger antigens. A number of strategies for antigen delivery by these strains have been attempted, ranging from plasmid-based to chromosomal integration systems. We report here the chromosomal integration of the T7 RNA polymerase gene (T7pol) in the attenuated strain Salmonella enterica serovar Typhi (Salmonella typhi) CVD908 (aroC(-), aroD(-)). The T7pol gene was amplified by PCR from Escherichia coli BL21(DE3) and cloned in the pNir3 plasmid under the control of the anaerobically inducible nirB promoter. Then it was subcloned in a pKTN701 derivative, suicide plasmid with the R6K ori, and flanked by the aroC gene. After evaluation of its functionality in E. coli SY327, the aroC-T7pol-aroC cassette was integrated into the aroC locus of S. typhi CVD908 by homologous recombination. The resulting strain, S. typhi CVD908-T7pol, was able to transcomplement two plasmids bearing the luc or the lacZ reporter genes controlled by the T7 promoter and produce luciferase and beta-galactosidase under anaerobic culture conditions. Therefore, an inducible system for recombinant antigen production in attenuated S. typhi was achieved.
Santiago-Machuca
Araceli E
AE
Unidad de Investigación Médica en Infectología e Inmunología, Hospital de Infectología, Dr. Daniel Méndez, La Raza, CMN, México City, México.
Ruiz-Pérez
Fernando
F
Delgado-Dominguez
José S
JS
Becker
Ingeborg
I
Isibasi
Armando
A
González-Bonilla
César R
CR
D001426:Bacterial Proteins / Q000235:genetics; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D009572:Nitrite Reductases; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012485:Salmonella typhi / Q000235:genetics*; D014613:Vaccines, Attenuated / Q000235:genetics; D014764:Viral Proteins
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D014613:Vaccines, Attenuated; D014764:Viral Proteins; D009572:Nitrite Reductases; C079730:NirB protein, E coli; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases