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: 2.3). 09/2004; 321(1):192-6. DOI: 10.1016/j.bbrc.2004.06.118
Source: PubMed


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.


Available from: Paulo Ho, Jul 27, 2015
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    • "The expression and purification of rFIM2 and CTB-Fim2 was performed as previously described for other recombinant proteins [27] [28]. Briefly, BL21(SI) E. coli competent cells were transformed with the pDEST17-fim2 or pAE-ctxB-fim2 plasmids and grown overnight (ON) at 37 ∘ C. Ampicillinresistant colonies were inoculated in 5 mL on Luria Bertani (LB) medium with ampicillin (50 í µí¼‡g/mL) without NaCl and grown ON at 37 ∘ C. On the following day, cultures were diluted 50-fold in LB-amp without NaCl and grown until A 600 reached 0.8, when NaCl was added to the medium at a final concentration of 300 mM. "
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    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
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    • "Image J software ( was used to analyze the bands in digital photographs of the gels to determine the percentage of CTB pentamer in HHP-treated samples in comparison to the total amount of CTB in IB. 2.6. ELISA for the determination of the binding of CTB to the GM1 receptor The ability of the CTB pentamers to bind to the GM1 receptor was assessed by ELISA, as described (Areas et al., 2004). Briefly, 96- well plates were coated with 2 ␮g/ml GM1 ganglioside in PBS, pH 7.2, or BSA in 0.05 mol/L carbonate–bicarbonate buffer, pH 9.6 at 37 • C for 4 h. "
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    ABSTRACT: The production of recombinant proteins is an essential tool for the expansion of modern biological research and biotechnology. The expression of heterologous proteins in E. coli often results in an incomplete folding process that leads to the accumulation of inclusion bodies (IB), aggregates that hold a certain degree of native-like secondary structure. High hydrostatic pressure (HHP) impairs intermolecular hydrophobic and electrostatic interactions, leading to dissociation of aggregates under non-denaturing conditions and is therefore a useful tool to solubilize proteins for posterior refolding. Cholera toxin (CT) is composed of a non-toxic pentamer of B subunits (CTB), a useful adjuvant in vaccines, and a toxic subunit A (CTA). We studied the process of refolding of CTB using HHP. HHP was shown to be effective for dissociation of CTB monomers from IB. Posterior incubation at atmospheric pressure of concentrated CTB (1mg/ml) is necessary for the association of the monomers. Pentameric CTB was obtained when suspensions of CTB IB were compressed at 2.4kbar for 16hours in the presence of Tween 20 and incubated at 1bar for 120h. Soluble and biologically active pentameric CTB was obtained, with a yield of 213mg CTB/liter of culture. The experience gained in this study can be important to improve the refolding of proteins with quaternary structure.
    Journal of Biotechnology 01/2014; 173(1). DOI:10.1016/j.jbiotec.2013.12.006 · 2.87 Impact Factor
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    • "This is now believed that CTB is effective oral immunogen that can stimulate mucosal immunity and create protection against cholera and entrotoxigenic E. coli infections (Sanchez and Holmgren 1986). According to the different studies by investigators it has been proven that CTB is a potent immunogen on nasal and mucosal system (Sun et al. 1994; Rudin et al. 1999; Areˆas et al. 2004; D'Ambrosio et al. 2008). Peru-15pCTB, attempt 1.3 (VA1.3) and IEM108 were examples of vaccines developed against cholera through genetic manipulation of domestic or standard strains to produce higher amounts of CTB and reduced zero level of CTA (Thungapathra et al. 1999; Fontana et al. 2001; Liang et al. 2003; Roland et al. 2007). "
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    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 l(-1) . GM(1) -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 titre 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 strains, made it a potential candidate for an indigenous vaccine. Significance and impact of the study: It was proved that rCTB produced in this system can be used as a potent immunogenic 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.
    Journal of Applied Microbiology 10/2012; 114(2). DOI:10.1111/jam.12043 · 2.48 Impact Factor
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