Expression and characterization of cholera toxin B-pneumococcal surface adhesin A fusion protein in Escherichia coli: ability of CTB-PsaA to induce humoral immune response in mice.

Centro de Biotecnologia, Instituto Butantan, São Paulo, Brazil.
Biochemical and Biophysical Research Communications (Impact Factor: 2.28). 09/2004; 321(1):192-6. DOI: 10.1016/j.bbrc.2004.06.118
Source: PubMed

ABSTRACT Cholera toxin B subunit (CTB) is responsible for CT holotoxin binding to the cell and has been described as a mucosal adjuvant for vaccines. In this work, the ctxB gene was genetically fused to the psaA gene from Streptococcus pneumoniae, a surface protein involved in its colonization in the host that is also considered a vaccine antigen candidate against this pathogen. The CTB-PsaA fusion protein was expressed in Escherichia coli, and the purified protein was used for intranasal immunization experiments in Balb/C mice. CTB-PsaA was able to induce both systemic and mucosal antibodies evaluated in serum, saliva, and in nasal and bronchial wash samples, showing that CTB-PsaA is a promising molecule to be investigated as S. pneumoniae vaccine antigen candidate.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: This study examined the immunogenic properties of the fusion protein fimbria 2 of Bordetella pertussis (Fim2)—cholera toxin B subunit (CTB) in the intranasal murine model of infection. To this end B. pertussis Fim2 coding sequence was cloned downstream of the cholera toxin B subunit coding sequence. The expression and assembly of the fusion protein into pentameric structures (CTB- Fim2) were evaluated by SDS-PAGE and monosialotetrahexosylgaglioside (GM1-ganglioside) enzyme-linked immunosorbent assay (ELISA). To evaluate the protective capacity of CTB-Fim2, an intraperitoneal or intranasal mouse immunization schedule was performed with 50 𝜇g of CTB-Fim2. Recombinant (rFim2) or purified (BpFim2) Fim2, CTB, and phosphate-buffered saline (PBS) were used as controls. The results showed that mice immunized with BpFim2 or CTB-Fim2 intraperitoneally or intranasally presented a significant reduction in bacterial lung counts compared to control groups (𝑃 < 0.01 or 𝑃 < 0.001, resp.). Moreover, intranasal immunization with CTB-Fim2 induced significant levels of Fim2-specific IgG in serum and bronchoalveolar lavage (BAL) and Fim2-specific IgA in BAL. Analysis of IgG isotypes and cytokines mRNA levels showed that CTB-Fim2 results in a mixed Th1/Th2 (T-helper) response. The data presented here provide support for CTB-Fim2 as a promising recombinant antigen against Bordetella pertussis infection.
    BioMed Research International 05/2014; 2014(Article ID 421486). DOI:10.1155/2014/421486 · 2.71 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: AIM: The aim of this study was to express and purify the recombinant CTB (rCTB) protein from Vibrio cholerae and investigate the biological and immunological characteristics of purified protein in rabbit animal model and in combination with Iranian inactivated V. cholerae whole cells as a domestic recombinant WC-CTB vaccine. METHODS AND RESULTS: Expressed 6XHis-tagged rCTB was properly purified, and its identity was confirmed by western blotting using cholera toxin-specific antibody. Concentration of purified protein was assessed to be 700 mg/lit. GM1-ELISA assay showed that purified rCTB pentamer was functionally active and able to bind GM(1) in a dose-dependent manner. Recombinant CTB was inoculated into rabbits through intestinal rout alone and in combination with inactivated whole-cell V. cholerae strains (WC). The anti-CTB IgG titer showed that Serum IgG responses were significantly increased in groups immunized with rCTB mixed with inactivated WC in comparison with control group. Furthermore, rCTB without V. cholerae WC also stimulated the IgG responses when inoculated into rabbit intestine. Challenge experiments of immunized rabbits showed an adequate protection against V. cholerae strains. CONCLUSIONS: Recombinant CTB alone and in combination with inactivated Iranian strains was protective against live toxigenic V.cholerae starins, made it a potential candidate for an indigenous vaccine. SIGNIFICANCE AND IMPACT OF STUDY: It was proved that rCTB produced in this system can be used as a potent immunogen protein to stimulate the immunity against V. cholerae strains and can be used for developing a native vaccine composed of our local strains with their own surface structures and antigenic determinants against cholera. © 2012The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.
    Journal of Applied Microbiology 10/2012; 114(2). DOI:10.1111/jam.12043 · 2.39 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cholera toxin B subunit (CtxB) is a homopantameric, nontoxic subunit of cholera toxin that is responsible for its binding to the cell and has been known as a mucosal adjuvant for vaccines that could increase homoral and mocusal immunity response. In this work, the CtxB gene was fused to the StxB gene from Shigella dysenteriae type I a vaccine antigen candidate against this pathogen, by a nonfurin linker then ligated with pGEM vector and subcloned in the pET28a(+) as an expression vector . The CtxB-StxB fusion protein was expressed in Escherichia coli, and purified by a Ni-NTA resin column, then detected molecular weight and immunogenicity by SDS-PAGE and Western-blot. StxB has low molecular weight, so immune response against it is low, while CtxB is a potent mucosal adjuvant. In this method, the CtxB-StxB fusion protein was expressed in Escherichia coli in order to use its natural adjuvanticity of the CtxB which will enhance immune response against StxB, as well as, it will produce immune response against both shiga and cholera toxins.