摘要
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/106 splenocytes） was higher than that induced by non-adjuvanted （（520±150） spot forming cells/10e splenocytes）, all responses against different antigens were enhanced in parallel. Conclusion CTB could be efficiently expressed in the presence of chloramphenicol and purified CTB is functional and capable of enhancincl the specific T cell responses elicited by DNA vaccine, the mechanism needs to be explored in the future.
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/106 splenocytes） was higher than that induced by non-adjuvanted （（520±150） spot forming cells/10e splenocytes）, all responses against different antigens were enhanced in parallel. Conclusion CTB could be efficiently expressed in the presence of chloramphenicol and purified CTB is functional and capable of enhancincl the specific T cell responses elicited by DNA vaccine, the mechanism needs to be explored in the future.
基金
This study was supported by the grants from the Chinese National Grand Program on Key Infectious Disease Control （No. 2008ZX10001-012 and -002）, the National Natural Science Foundation of China （No. 81072496H1014） and the Fund for Young Scientists from Fudan University （No. 07L03）.
