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 18kDa protein was cl oned between the two copies of IS900 to provide expression signals. Co nstructs were introduced into Mycobacterium species smegmatis, vaccae and bovis BCG by electroporation and selection for kanamycin resistanc e. The expression of the 18 kDa gene was analysed by Western blotting. Integration of the vector into the M. smegmatis chromosome was analys ed by Southern blotting. One to five copies of the vector were detecte d in each transformant. The SIV gag p27 gene and the foot-and-mouth di sease virus VP1 140-160 epitope were successfully cloned into the 18 k Da gene and expression in M. smegmatis was obtained.