Cloning and constitutive expression of structural genes encoding gonococcal porin protein in Escherichia coli and attenuated Salmonella typhimurium vaccine strains.
Previous reports [Gotschlich et al., Proc. Natl. Acad. Sci. USA 84 (1987) 8135-8139; Carbonetti and Sparling, Proc. Natl. Acad. Sci. USA 84 (1987) 9084-9088; Carbonetti et al., Proc. Natl. Acad. Sci. USA 85 (1988) 6841-6845] concluded that synthesis of the porin protein (Por) from Neisseria gonorrhoeae in Escherichia coli was toxic to that organism, which limited studies of the biology of Por in foreign hosts. We assembled intact por genes from the gonococcal strains, FA19 (serogroup PIA) and FA6434 (a hybrid Por containing epitopes from serogroups PIA and PIB), and observed stable expression in E. coli without evident toxicity. Expression of por from strain MS11 (serogroup PIB) in E. coli was difficult, but por from MS11 was expressed without toxicity when the -35 region of the por promoter was removed. Encouraged by this, we moved por from E. coli into attenuated Salmonella typhimurium strains and expressed por either in single copy from the chromosome or in multiple copy from plasmids. Expression levels of por in S. typhimurium were higher from plasmids than from the chromosome, probably due to a gene dosage effect. This work will enable study of the immune response to Por in mice vaccinated orally with live S. typhimurium.
Elkins
C
C
Department of Medicine, University of North Carolina at Chapel Hill 27599.
Carbonetti
N H
NH
Coímbre
A J
AJ
Thomas
C E
CE
Sparling
P F
PF
D001428:Bacterial Vaccines; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism*; D005798:Genes, Bacterial; D005838:Genotype; D008969:Molecular Sequence Data; D009344:Neisseria gonorrhoeae / Q000235:genetics* / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D010641:Phenotype; D010957:Plasmids; D018272:Porins / Q000096:biosynthesis* / Q000235:genetics; D015183:Restriction Mapping; D012486:Salmonella typhimurium / Q000378:metabolism*; D014613:Vaccines, Attenuated
D001428:Bacterial Vaccines; D009838:Oligodeoxyribonucleotides; D018272:Porins; D014613:Vaccines, Attenuated