Construction and use of integrative vectors to express foreign genes in mycobacteria

Abstract

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.

IS900 targets translation initiation signals in Mycobacterium avium subsp. paratuberculosis to facilitate expression of its hed gene

“…AS IS900 has been utilized for the construction of integrative vectors for stable expression of foreign genes in mycobacteria (England et al, 1991 ;Dellagostin et al, 1993), clarification of the mechanism of IS900 transposition may increase its potential and applications as a genetic tool for the manipulation of mycobacteria. A new application may be insertional mutagenesis for investigation of virulence mechanisms in pathogenic mycobacteria.…”

Plasmid instability when the <i>hsp</i>60 gene promoter is used to express the protective non-toxic fragment B of the diphtheria toxin in recombinant BCG

“…The genetic modification of the BCG vaccine as an approach to the development of new multivalent vaccines has been actively pursued ever since it was proposed in 1989 [8] and has been the focus of our research over the last years [21][22][23][24][25][26][27][28]. BCG is a powerful candidate to a multivalent vaccine platform.…”

Context-sensitive transposition of IS6110 in mycobacteria

“…The mycobacterial strains used were Mycobacterium smegmatis mc#155 (Snapper et al, 1990) and Mycobacterium bovis BCG (Pasteur strain). Electroporation of mycobacteria was by previously described methods (Dellagostin et al, 1993 . IS, IS6110 (PCR-amplified using the primer of Fomukong & Dale, 1993) ; aph, kanamycin-resistance gene ; T7, T7 promoter ; P, PvuII.…”