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. 1989 Jan;86(2):481–485. doi: 10.1073/pnas.86.2.481

Recombinant system for overexpression of cholera toxin B subunit in Vibrio cholerae as a basis for vaccine development.

J Sanchez 1, J Holmgren 1
PMCID: PMC286494  PMID: 2492108

Abstract

We have constructed an overexpression system in which the gene encoding the B subunit of cholera toxin (CTB) was placed under the control of the strong tacP promoter in a wide host range plasmid. Recombinant nontoxigenic classical and E1 Tor Vibrio cholerae strains of different serotypes harboring this plasmid excreted 10- to 100-fold higher amounts of CTB than any other wild-type or recombinant strain tested and may therefore be useful killed oral vaccine strains. The manipulations to place the CTB gene under tacP also included, by design, the introduction of single enzyme restriction sites for gene fusions to the CTB amino terminus. Cloning into these sites allows construction of CTB-derived hybrid proteins carrying various putative vaccine peptide antigens.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Clemens J. D., Sack D. A., Harris J. R., Chakraborty J., Khan M. R., Stanton B. F., Kay B. A., Khan M. U., Yunus M., Atkinson W. Field trial of oral cholera vaccines in Bangladesh. Lancet. 1986 Jul 19;2(8499):124–127. doi: 10.1016/s0140-6736(86)91944-6. [DOI] [PubMed] [Google Scholar]
  2. Clemens J. D., Sack D. A., Harris J. R., Chakraborty J., Neogy P. K., Stanton B., Huda N., Khan M. U., Kay B. A., Khan M. R. Cross-protection by B subunit-whole cell cholera vaccine against diarrhea associated with heat-labile toxin-producing enterotoxigenic Escherichia coli: results of a large-scale field trial. J Infect Dis. 1988 Aug;158(2):372–377. doi: 10.1093/infdis/158.2.372. [DOI] [PubMed] [Google Scholar]
  3. Craig J. P. A permeability factor (toxin) found in cholera stools and culture filtrates and its neutralization by convalescent cholera sera. Nature. 1965 Aug 7;207(997):614–616. doi: 10.1038/207614a0. [DOI] [PubMed] [Google Scholar]
  4. Donta S. T., Damiano-Burbach P., Poindexter N. J. Modulation of enterotoxin binding and function in vitro and in vivo. J Infect Dis. 1988 Mar;157(3):557–564. doi: 10.1093/infdis/157.3.557. [DOI] [PubMed] [Google Scholar]
  5. Fürste J. P., Pansegrau W., Frank R., Blöcker H., Scholz P., Bagdasarian M., Lanka E. Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene. 1986;48(1):119–131. doi: 10.1016/0378-1119(86)90358-6. [DOI] [PubMed] [Google Scholar]
  6. Gennaro M. L., Greenaway P. J. Nucleotide sequences within the cholera toxin operon. Nucleic Acids Res. 1983 Jun 25;11(12):3855–3861. doi: 10.1093/nar/11.12.3855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Glass R. I., Holmgren J., Khan M. R., Hossain K. M., Huq M. I., Greenough W. B. A randomized, controlled trial of the toxin-blocking effects of B subunit in family members of patients with cholera. J Infect Dis. 1984 Apr;149(4):495–500. doi: 10.1093/infdis/149.4.495. [DOI] [PubMed] [Google Scholar]
  8. Hardy S. J., Holmgren J., Johansson S., Sanchez J., Hirst T. R. Coordinated assembly of multisubunit proteins: oligomerization of bacterial enterotoxins in vivo and in vitro. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7109–7113. doi: 10.1073/pnas.85.19.7109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Holmgren J. Actions of cholera toxin and the prevention and treatment of cholera. Nature. 1981 Jul 30;292(5822):413–417. doi: 10.1038/292413a0. [DOI] [PubMed] [Google Scholar]
  10. Holmgren J., Fredman P., Lindblad M., Svennerholm A. M., Svennerholm L. Rabbit intestinal glycoprotein receptor for Escherichia coli heat-labile enterotoxin lacking affinity for cholera toxin. Infect Immun. 1982 Nov;38(2):424–433. doi: 10.1128/iai.38.2.424-433.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jertborn M., Svennerholm A. M., Holmgren J. Five-year immunologic memory in Swedish volunteers after oral cholera vaccination. J Infect Dis. 1988 Feb;157(2):374–377. doi: 10.1093/infdis/157.2.374. [DOI] [PubMed] [Google Scholar]
  12. Kaper J. B., Lockman H., Baldini M. M., Levine M. M. Recombinant nontoxinogenic Vibrio cholerae strains as attenuated cholera vaccine candidates. Nature. 1984 Apr 12;308(5960):655–658. doi: 10.1038/308655a0. [DOI] [PubMed] [Google Scholar]
  13. Klipstein F. A., Engert R. F., Houghten R. A. Protection in rabbits immunized with a vaccine of Escherichia coli heat-stable toxin cross-linked to the heat-labile toxin B subunit. Infect Immun. 1983 Jun;40(3):888–893. doi: 10.1128/iai.40.3.888-893.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kurosky A., Markel D. E., Peterson J. W. Covalent structure of the beta chain of cholera enterotoxin. J Biol Chem. 1977 Oct 25;252(20):7257–7264. [PubMed] [Google Scholar]
  15. Lai C. Y. Determination of the primary structure of cholera toxin B subunit. J Biol Chem. 1977 Oct 25;252(20):7249–7256. [PubMed] [Google Scholar]
  16. Leong J., Vinal A. C., Dallas W. S. Nucleotide sequence comparison between heat-labile toxin B-subunit cistrons from Escherichia coli of human and porcine origin. Infect Immun. 1985 Apr;48(1):73–77. doi: 10.1128/iai.48.1.73-77.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lindholm L., Holmgren J., Wikström M., Karlsson U., Andersson K., Lycke N. Monoclonal antibodies to cholera toxin with special reference to cross-reactions with Escherichia coli heat-labile enterotoxin. Infect Immun. 1983 May;40(2):570–576. doi: 10.1128/iai.40.2.570-576.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lockman H., Kaper J. B. Nucleotide sequence analysis of the A2 and B subunits of Vibrio cholerae enterotoxin. J Biol Chem. 1983 Nov 25;258(22):13722–13726. [PubMed] [Google Scholar]
  19. McClure W. R. Mechanism and control of transcription initiation in prokaryotes. Annu Rev Biochem. 1985;54:171–204. doi: 10.1146/annurev.bi.54.070185.001131. [DOI] [PubMed] [Google Scholar]
  20. McKenzie S. J., Halsey J. F. Cholera toxin B subunit as a carrier protein to stimulate a mucosal immune response. J Immunol. 1984 Oct;133(4):1818–1824. [PubMed] [Google Scholar]
  21. Mekalanos J. J., Swartz D. J., Pearson G. D., Harford N., Groyne F., de Wilde M. Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development. Nature. 1983 Dec 8;306(5943):551–557. doi: 10.1038/306551a0. [DOI] [PubMed] [Google Scholar]
  22. Sanchez J., Hirst T. R., Uhlin B. E. Hybrid enterotoxin LTA::STa proteins and their protection from degradation by in vivo association with B-subunits of Escherichia coli heat-labile enterotoxin. Gene. 1988 Apr 29;64(2):265–275. doi: 10.1016/0378-1119(88)90341-1. [DOI] [PubMed] [Google Scholar]
  23. Sanchez J., Svennerholm A. M., Holmgren J. Genetic fusion of a non-toxic heat-stable enterotoxin-related decapeptide antigen to cholera toxin B-subunit. FEBS Lett. 1988 Dec 5;241(1-2):110–114. doi: 10.1016/0014-5793(88)81041-x. [DOI] [PubMed] [Google Scholar]
  24. Sanchez J., Uhlin B. E., Grundström T., Holmgren J., Hirst T. R. Immunoactive chimeric ST-LT enterotoxins of Escherichia coli generated by in vitro gene fusion. FEBS Lett. 1986 Nov 24;208(2):194–198. doi: 10.1016/0014-5793(86)81016-x. [DOI] [PubMed] [Google Scholar]
  25. Sandkvist M., Hirst T. R., Bagdasarian M. Alterations at the carboxyl terminus change assembly and secretion properties of the B subunit of Escherichia coli heat-labile enterotoxin. J Bacteriol. 1987 Oct;169(10):4570–4576. doi: 10.1128/jb.169.10.4570-4576.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Svennerholm A. M., Wikström M., Lindblad M., Holmgren J. Monoclonal antibodies against Escherichia coli heat-stable toxin (STa) and their use in a diagnostic ST ganglioside GM1-enzyme-linked immunosorbent assay. J Clin Microbiol. 1986 Oct;24(4):585–590. doi: 10.1128/jcm.24.4.585-590.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tayot J. L., Holmgren J., Svennerholm L., Lindblad M., Tardy M. Receptor-specific large-scale purification of cholera toxin on silica beads derivatized with lysoGM1 ganglioside. Eur J Biochem. 1981 Jan;113(2):249–258. doi: 10.1111/j.1432-1033.1981.tb05060.x. [DOI] [PubMed] [Google Scholar]
  30. WADSWORTH C., HANSON L. A. Comparative analysis of immune electrophoretic precipitates employing a modified immune electrophoretic technique. Int Arch Allergy Appl Immunol. 1960;17:165–177. doi: 10.1159/000229122. [DOI] [PubMed] [Google Scholar]
  31. Yamamoto T., Suyama A., Mori N., Yokota T., Wada A. Gene expression in the polycistronic operons of Escherichia coli heat-labile toxin and cholera toxin: a new model of translational control. FEBS Lett. 1985 Feb 25;181(2):377–380. doi: 10.1016/0014-5793(85)80296-9. [DOI] [PubMed] [Google Scholar]
  32. de Aizpurua H. J., Russell-Jones G. J. Oral vaccination. Identification of classes of proteins that provoke an immune response upon oral feeding. J Exp Med. 1988 Feb 1;167(2):440–451. doi: 10.1084/jem.167.2.440. [DOI] [PMC free article] [PubMed] [Google Scholar]

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