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Chloramphenicol improved expression of recombinant cholera toxin B subunit in Escherichia coli and its adjuvanticity / 中华医学杂志(英文版)
Chinese Medical Journal ; (24): 2751-2755, 2011.
Article Dans Anglais | WPRIM | ID: wpr-292811
ABSTRACT
<p><b>BACKGROUND</b>Cholera toxin B subunit (CTB) was shown to be a potent adjuvant for protein immunogen, especially when inoculated through mucosal route. We aimed to optimize the expression approach for CTB and thereafter to determine the adjuvant effect on DNA vaccine.</p><p><b>METHODS</b>Wild type CTB coding gene was amplified and cloned into prokaryotic expression vector pET-30a, and the recombinant CTB was expressed in the presence of different concentration of chloramphenicol and isopropyl β-D-thiogalactoside. Purified recombinant CTB was mixed with HIV-1 AE2f tat-rev-integrase-vif-nef fusion gene DNA vaccine and female BALB/c mice were vaccinated with a DNA priming-recombinant vaccinia vectored vaccine boosting regimen through intramuscular injection. Interferon γ (IFN-γ) enzyme-linked immunospot (Elispot) assay was used to read out the specific T-cell immunity.</p><p><b>RESULTS</b>Chloramphenicol was essential for the efficient expression of recombinant CTB (rCTB) in pET-30a/BL21 (DE3) system and could be optimized at the concentration of 0.625 µg/ml in the presence of chloramphenicol. The purified rCTB could bind with GM1 efficiently. INF-γ Elispot data showed the T-cell response induced in CTB adjuvanted group ((734 ± 240) spot forming cells/10(6) splenocytes) was higher than that induced by non-adjuvanted ((520 ± 150) spot forming cells/10(6) splenocytes), all responses against different antigens were enhanced in parallel.</p><p><b>CONCLUSION</b>CTB could be efficiently expressed in the presence of chloramphenicol and purified CTB is functional and capable of enhancing the specific T cell responses elicited by DNA vaccine, the mechanism needs to be explored in the future.</p>
Sujets)
Texte intégral: Disponible Indice: WPRIM (Pacifique occidental) Sujet Principal: Pharmacologie / Protéines de fusion recombinantes / Chloramphénicol / Adjuvants immunologiques / Toxine cholérique / Technique de Western / Vaccins à ADN / Électrophorèse sur gel de polyacrylamide / Allergie et immunologie / Escherichia coli Limites du sujet: Animaux langue: Anglais Texte intégral: Chinese Medical Journal Année: 2011 Type: Article

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Texte intégral: Disponible Indice: WPRIM (Pacifique occidental) Sujet Principal: Pharmacologie / Protéines de fusion recombinantes / Chloramphénicol / Adjuvants immunologiques / Toxine cholérique / Technique de Western / Vaccins à ADN / Électrophorèse sur gel de polyacrylamide / Allergie et immunologie / Escherichia coli Limites du sujet: Animaux langue: Anglais Texte intégral: Chinese Medical Journal Année: 2011 Type: Article