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...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.展开更多
Neural tract tracing is used to study neural pathways and evaluate neuronal regeneration following nerve injuries.However,it is not always clear which tracer should be used to yield optimal results.In this study,we ex...Neural tract tracing is used to study neural pathways and evaluate neuronal regeneration following nerve injuries.However,it is not always clear which tracer should be used to yield optimal results.In this study,we examined the use of Alexa Fluor 488-conjugated cholera toxin subunit B(AF488-CTB).This was injected into the gastrocnemius muscle of rats,and it was found that motor,sensory,and sympathetic neurons were labeled in the spinal ventral horn,dorsal root ganglia,and sympathetic chain,respectively.Similar results were obtained when we injected AF594-CTB into the tibialis anterior muscle.The morphology and number of neurons were evaluated at different time points following the AF488-CTB injection.It was found that labeled motor and sensory neurons could be observed 12 hours post-injection.The intensity was found to increase over time,and the morphology appeared clear and complete 3-7 days post-injection,with clearly distinguishable motor neuron axons and dendrites.However,14 days after the injection,the quality of the images decreased and the neurons appeared blurred and incomplete.Nissl and immunohistochemical staining showed that the AF488-CTB-labeled neurons retained normal neurochemical and morphological features,and the surrounding microglia were also found to be unaltered.Overall,these results imply that the cholera toxin subunit B,whether unconjugated or conjugated with Alexa Fluor,is effective for retrograde tracing in muscular tissues and that it would also be suitable for evaluating the regeneration or degeneration of injured nerves.展开更多
目的构建霍乱弧菌肠毒素B亚单位(Cholera toxin B subunit,CTB)基因的大肠杆菌表达重组质粒,并观察其在大肠杆菌和双歧杆菌中的表达。方法从pBI121质粒PCR扩增获得CTB基因片断,克隆到大肠杆菌载体pGEX-4T-1上,构建重组质粒,然后转化大...目的构建霍乱弧菌肠毒素B亚单位(Cholera toxin B subunit,CTB)基因的大肠杆菌表达重组质粒,并观察其在大肠杆菌和双歧杆菌中的表达。方法从pBI121质粒PCR扩增获得CTB基因片断,克隆到大肠杆菌载体pGEX-4T-1上,构建重组质粒,然后转化大肠杆菌DH5α和双歧杆菌。转化菌经IPTG诱导,然后用SDS-PAGE和Western blot方法鉴定表达的重组蛋白。结果构建了重组质粒pGEX-4T-CTB,CTB基因片段分子量约为376bp;在大肠杆菌中表达出35kD的霍乱弧菌B亚单位融合蛋白,经SDS-PAGE分析,相对分子量与文献相符,表达的蛋白约占细菌总蛋白的10%;在双岐杆菌中也能得到正确表达,表达量较大肠杆菌低,占细菌总蛋白约5%。Western blotting结果确认了该条带为CTB基因的产物。结论构建的重组质粒pGEX-4T-CTB能够在大肠杆菌及双歧杆菌中获得表达。展开更多
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）.
文摘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supported by the CACMS Innovation Fund,No.CI2021A03407(to WZB)the Project of National Key R&D Program of China,No.2019YFC1709103(to WZB)+1 种基金the National Natural Science Foundation of China,Nos.81774432(to JJC),81774211(to WZB),82004492(to JW),81801561(to DSX)the Fundamental Research Funds for the Central Public Welfare Research Institutes of China,Nos.ZZ13-YQ-068(to JJC),ZZ14-YQ-032(to JW),ZZ14-YQ-034(to DSX).
文摘Neural tract tracing is used to study neural pathways and evaluate neuronal regeneration following nerve injuries.However,it is not always clear which tracer should be used to yield optimal results.In this study,we examined the use of Alexa Fluor 488-conjugated cholera toxin subunit B(AF488-CTB).This was injected into the gastrocnemius muscle of rats,and it was found that motor,sensory,and sympathetic neurons were labeled in the spinal ventral horn,dorsal root ganglia,and sympathetic chain,respectively.Similar results were obtained when we injected AF594-CTB into the tibialis anterior muscle.The morphology and number of neurons were evaluated at different time points following the AF488-CTB injection.It was found that labeled motor and sensory neurons could be observed 12 hours post-injection.The intensity was found to increase over time,and the morphology appeared clear and complete 3-7 days post-injection,with clearly distinguishable motor neuron axons and dendrites.However,14 days after the injection,the quality of the images decreased and the neurons appeared blurred and incomplete.Nissl and immunohistochemical staining showed that the AF488-CTB-labeled neurons retained normal neurochemical and morphological features,and the surrounding microglia were also found to be unaltered.Overall,these results imply that the cholera toxin subunit B,whether unconjugated or conjugated with Alexa Fluor,is effective for retrograde tracing in muscular tissues and that it would also be suitable for evaluating the regeneration or degeneration of injured nerves.
