Abstract
Gene fusion proteins with epitopes attached to the amino end of cholera toxin B subunit (CTB) are useful to raise immunological responses. We describe a cloning vector, designated pCTBtet, carrying a tetracycline resistance gene (TetR) between the leader peptide and mature CTB. Removal of TetR to insert oligonucleotides encoding fusion epitopes allowed for screening of tetracycline-sensitive clones. Restoration of the correct CTB reading phase was subsequently used to choose gene fusion candidate colonies. The use of pCTBtet permitted the rapid construction of 8 fusion proteins carrying 9–24 aa fromSalmonella typhi OmpC and 6 hybrids with 7–31 aa fromEscherichia coli colonization factor CFAI.
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References
Elson, C. O. (1997) In defense of mucosal surfaces. Regulation and manipulation of the mucosal immune system.Adv. Exp. Med. Biol. 412, 373–385.
Holmgren, J. and Czerkinsky, C. (1992) Cholera as a model for research on mucosal immunity and development of oral vaccines.Curr. Opinion Immunol. 4, 387–391.
Sun, J-B., Rask, C., Olsson, T., Holmgren, J., and Czerkinsky, C. (1996) Treatment of experimental autoimmune encephalomyelitis by feeding myelin basic protein conjugated to cholera toxin B subunit.Proc. Natl. Acad. Sci. USA 93, 7196–7201.
Bergerot, Y., Ploix, C., Petersen, J., Moulin, V., Rask, C., Fabien, N., et al. (1997) A cholera toxoid-insulin conjugate as an oral vaccine against spontaneous autoimmune diabetes.Proc. Natl. Acad. Sci. USA 94, 4610–4614.
Sanchez, J., Svennerholm, A. M., and Holmgren, J. (1988) Genetic fusion of a non-toxic heat-stable enterotoxin-related decapeptide antigen to cholera toxin B subunit.FEBS Lett. 241, 110–114.
González, R., Sanchez, J., Holmgren, J., López, S., and Arias, C. (1993) Immunological characterization of a rotavirus-neutralizing epitope fused to the cholera toxin B subunit.Gene 133, 227–232.
Laloi, P., Munro, C. L. Jones, K. R., and Macrina, F. L. (1996) Immunological characteristics of aStreptococcus mutans glucosyltransferase B sucrose-binding site peptide-cholera toxin B-subunit chimeric protein.Infect. Immun. 64, 28–36.
Ryan, E. T., Butterton, J. R., Zhang, T., Baker, M. A., Stanley, S. L., Jr., and Calderwood, S. B. (1997) Oral immunization with attenuated vaccine strains ofVibrio cholerae expressing a dodecapeptide repeat of the serine-richEntamoeba histolytica protein fused to the cholera toxin B subunit induces systemic and mucosal antiamebic and anti-V.cholerae antibody responses in mice.Infect. Immun. 65, 3118–3125.
Paniagua-Solis, J., Sanchez, J., Ortiz, V., Gonzalez, C., and Isibasi, A. (1996) Construction of CTB fusion proteins for screening of monoclonal antibodies againstSalmonella typhi OmpC peptide loops.FEMS Microbiol. Lett. 141, 31–36
Sanchez, J., Johansson, S., Lowenadler, B., Svennerholm, A. M., and Holmgren, J. (1990) Recombinant cholera toxin B subunit and gene fusion proteins for oral vaccination.Res. Microbiol. 141, 971–979.
BÄckström, M., Holmgren, J., Schödel, F., and Lebens, M. (1995) Characterization of an internal permissive site in the cholera toxin B-subunit and insertion of epitopes from human immunodeficiency virus-1, hepatitis B virus and enterotoxigenicEscherichia coli.Gene 165, 163–171.
L’hoir, C., Renard, A., and Martial, J. A. (1990) Expression inEscherichia coli of two mutated genes encoding the cholera toxin B subunit.Gene 89, 47–52.
Bolivar, F., Rodríguez, R. L., Greene, P. J., Betlach, M. C., Heyneke, H. L., and Boyer, H. W. (1977) Construction and characterization of new cloning vehicles II. A multipurpose cloning system.Gene 2, 95–113.
Cassels, F. J., Jarboe, D. L., Reid, R. H., Lees, A., and Deal, C. D. (1997) Linear epitopes of colonization factor antigen I and peptide vaccine approach to enterotoxigenicEscherichia coli.J. Ind. Microbiol. Biotechnol. 19, 66–70.
Svennerholm, A. M. and Holmgren, J. (1978) Identification ofEscherichia coli heat labile enterotoxin by means of a ganglioside immunoadsorbent assay (GM1-ELISA).Curr. Microbiol. 1, 19–23.
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Secundino, I., Paniagua-Solís, J., Isibasi, A. et al. A cloning vector for efficient generation of cholera toxin B gene fusions for epitope screening. Mol Biotechnol 11, 101–104 (1999). https://doi.org/10.1007/BF02789180
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DOI: https://doi.org/10.1007/BF02789180