Expression of human lactoferrin in Bacteroides uniformis and its effect on Azoxymethane-induced aberrant crypt Focus formation in the rat colon

Abstract

To express a human lactoferrin (hLF) gene in Bacteroides uniformis, a member of the anaerobic microflora in the colon, we constructed a recombinant plasmid, pVLFK, by subcloning hLF cDNA to an Escherichia coli-Bacteroides shuttle vector, pVAL-1. The plasmid pVLFK was transferred to B. uniformis strain BU1001 by the filter mating procedure creating strain TCTK101. The lactoferrin protein in B. uniformis strain TCTK101 was detected by Western blot analysis with an anti-human lactoferrin monoclonal antibody. A culture of strain TCTK101 inhibited the growth of E. coli strain HB101 in vitro compared to a culture of strain TCTK11, which is a B. uniformis straincarrying plasmid pVAL-1, suggesting that the lactoferrin produced from strain TCTK101 possesses biological activity. To determine the effect of lactoferrin-producing B. uniformis on the formation of azoxymethane-induced aberrant crypt foci (ACF), putative neoplastic lesions, overnight cultures of strains TCTK11 and TCTK101 were given to rats as drinking water. The numbers of ACF and ACF having more than three crypts per focus five weeks after the beginning of the experiment significantly increased in the group treated by a culture of strain TCTK11 compared with those in the non-treated water group. However, rats treated with a culture of strain TCTK101 carrying plasmid pVLFK showed a significantly lower number of ACF than rats with a culture of strain TCTK11 (34% reduction), suggesting that hLF which is produced in a prokaryotic expression system prevents formation of ACF in the rat colon.