Intracellular Expression of CTB in Strains in Laboratory Culture Conditions.

The introduction of the -139F allele triggers the expression of TCP (toxin co-regulated pilus) and CT (cholera toxin) under simple laboratory culture conditions in most strains. Such strains, especially strains that have been used in OCVs (oral cholera vaccines), can induce antibody responses against TCP in animal models. However, CT produced in these strains is secreted into the culture medium. In this study, strains that can express intracellular CTB under the control of the -139F allele have been constructed for potential application in OCVs. First, we constructed a recombinant plasmid directly linking the promoter to without and confirmed CTB expression from the plasmid in containing the -139F allele. We constructed another recombinant plasmid to express NtrCTB, from which 14 internal amino acids-from the 7 to the 20 amino acid-of the leader peptide of CTB have been omitted, and we found that NtrCTB remained in the cells. Based on those results, we constructed strains in which chromosomal is replaced by or -dimer. Both NtrCTB and NtrCTB-dimer remained in the bacterial cells, and 60% of the NtrCTB-dimer in the bacterial cells was maintained in a soluble form. To develop improved OCVs, these strains could be tested to see whether they induce immune responses against CTB in animal models.