title: Chloramphenicol improved expression of recombinant cholera toxin B subunit in Escherichia coli and its adjuvanticity.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: Shanghai Public Health Clinical Center, Institutes of Biomedical Sciences, Key Laboratory of Medical Molecular Virology of Ministry of Education, Shanghai Medical College, Fudan University, Shanghai 201508, China.
authors: Xiao-yan
authors: XY
authors: Xie
authors: Yan-min
authors: YM
authors: Wan
authors: Zhao-qin
authors: ZQ
authors: Zhu
authors: Huan-xiang
authors: HX
authors: Zhang
authors: Jian-qing
authors: JQ
authors: Xu
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002701:Chloramphenicol / Q000494:pharmacology*; D002772:Cholera Toxin / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects* / Q000378:metabolism*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D000276:Adjuvants, Immunologic; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; D002701:Chloramphenicol; D002772:Cholera Toxin