Construction and use of integrative vectors to express foreign genes in mycobacteria.
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.
Dellagostin
O A
OA
Molecular Microbiology Group, School of Biological Sciences, University of Surrey, Guildford, UK.
Wall
S
S
Norman
E
E
O'Shaughnessy
T
T
Dale
J W
JW
McFadden
J
J
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics; D005537:Aphthovirus / Q000235:genetics; D001483:Base Sequence; D002213:Capsid / Q000235:genetics; D036022:Capsid Proteins; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004251:DNA Transposable Elements; D004279:DNA, Viral / Q000235:genetics; D015870:Gene Expression; D015683:Gene Products, gag / Q000235:genetics; D005814:Genes, Viral; D015750:Genes, gag; D005822:Genetic Vectors; D007613:Kanamycin Resistance / Q000235:genetics; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics*; D009166:Mycobacterium leprae / Q000235:genetics / Q000276:immunology; D015302:Simian Immunodeficiency Virus / Q000235:genetics; D014170:Transformation, Genetic
D000942:Antigens, Bacterial; D036022:Capsid Proteins; D017931:DNA Primers; D004251:DNA Transposable Elements; D004279:DNA, Viral; C066594:Gag protein p27, Simian immunodeficiency virus; D015683:Gene Products, gag