Objective： To construct recombinant E.coli LLO/OVA and investigate its tumor metastatic inhibition effect in B16 OVA melanoma challenged mice. Methods： Recombinant E.coli LLO/OVA was constructed and the expression ...Objective： To construct recombinant E.coli LLO/OVA and investigate its tumor metastatic inhibition effect in B16 OVA melanoma challenged mice. Methods： Recombinant E.coli LLO/OVA was constructed and the expression of listeriolysin O （LLO） and ovalbumin （OVA） of the vaccine was determined by coomassie brilliant blue staining and western blotting, After 3 subcutaneous injections of E.coli LLO/OVA, the percentages of CD3^＋CD4^＋T, CD4^＋CD25^＋T, CD3^CD8^＋T and OVA257-264 SIINFEKL specific CD8^＋T cells were determined by flow cytomytry, and the tumor metastatic inhibition effect in B16 OVA melanoma challenged mice was observed. Results： Recombinant E.coli LLO/OVA was successfully constructed, and the expression of LLO and OVA of the vaccine was confirmed. After 3 subcutaneous injections of E.coli LLO/OVA and E.coli OVA in mice, the percentages of CD3^＋CD4^＋T, CD4^＋CD25^＋T and CD3^＋CD8^＋T cells were equivalent in the two groups of mice. However, there were significantly more OVA257-264 SIINFEKL specific CD8^＋T cells in E.coli LLO/OVA vaccinated mice than that in E.coli OVA vaccinated mice. The prophylactic E.coli LLO/OVA vaccination effectively prevented the tumor metastasis to lungs in B16 OVA melanoma challenged mice. Depletion of CD8^＋T cells significantly impaired the tumor inhibition effect of the vaccine in B16 OVA challenged mice. The therapeutic vaccination of E.coli LLO/OVA significantly prevented melanoma metastasis to lungs in B I6 OVA challenged mice too. Conclusion： Recombinant E.coli LLO/OVA vaccination is highly effective in inhibiting murine malignant melanoma metastasis by promoting CD8^＋T cell immunity.展开更多
[ Objective] To explore different preservation methods of recombinant E. coli and find out the optimal conditions for preservation. [ Method] The recombinant E. coli DH5cx transformed pcDNA.3 were respectively preserv...[ Objective] To explore different preservation methods of recombinant E. coli and find out the optimal conditions for preservation. [ Method] The recombinant E. coli DH5cx transformed pcDNA.3 were respectively preserved at 4℃ and -70 ℃, and the activity was determined after dif- ferent time. [ Result] The number of living E. coll with high dilutions preserved at 4 ℃ was gradually increased within the first 7 d, peaked on Day 7, and then gradually decreased. The number of living E. coli, which were preserved in 8% glycerol at -70℃ when OD800 at 0.8, were significantly higher than that of other groups after different preservation time. [ Conclusion] The optimal storage time was 7 d for recombinant E. coli at 4 ℃. For preservation at -70 ℃, the bacteria should be in logarithmic growth phase and preserved in 8% glycerol.展开更多
The gene encoding the 18 kDa protein of Taenia solium metacestodes was amplified by RT-PCR and cloned into the pGEM-T vector for sequencing.The recombinant plasmid named pGEX-CE18 was constructed and transformed into ...The gene encoding the 18 kDa protein of Taenia solium metacestodes was amplified by RT-PCR and cloned into the pGEM-T vector for sequencing.The recombinant plasmid named pGEX-CE18 was constructed and transformed into E.coli BL21 for in vitro expression.SDS-PAGE and Western blot were employed for analyzing the recombinant protein,which was then used for development of an indirect ELISA for detection of anti-cysticercosis antibodies.The results showed that the recombinant protein of interest was 35 kDa in size,accounting for 28%of total bacteria proteins,and reacted with positive sera against cysticercosis.Using the newly-constructed indirect ELISA and a commercially available ELISA kit,paired analyses of 178 serum samples indicated that the concordant rate was 98.83%and the ELISA exhibited good specificity and sensitivity,supporting its utility and application for diagnosis of cysticercosis.展开更多
The photosynthetic bacteria, Rodospirillum rubrum, produced hydrogen when grown in cheese whey in presence of light. The production increased three times as much when whey was used after being incubated in presence of...The photosynthetic bacteria, Rodospirillum rubrum, produced hydrogen when grown in cheese whey in presence of light. The production increased three times as much when whey was used after being incubated in presence of Escherichia coli at 37℃ for 5 days, giving a total of 3600 ml of H2 in 10 days. The presence of Fe ions (5 mg/L) enhanced H2 production of treated whey to about 6000 ml in 10 days. Mo ions (0.3 and 1.6 mg/l) did not affect achieved H2 production of treated whey. However, when Fe and Mo ions were present together, the production was comparable with that of Mo ions alone, i.e. Mo prevented Fe of producing any enhancing effect. The addition of Mn ions (7.68 mg/L) to treated whey containing Fe (5 mg/L) and Mo ions (8 mg/L) increased H2 production to give about 9500 ml/10 days.展开更多
基金supported by the State Scholarship Fund from China Scholarship Council (No.2003850064)Chongqing Educational Committee Foundation (cq20070319).
文摘Objective： To construct recombinant E.coli LLO/OVA and investigate its tumor metastatic inhibition effect in B16 OVA melanoma challenged mice. Methods： Recombinant E.coli LLO/OVA was constructed and the expression of listeriolysin O （LLO） and ovalbumin （OVA） of the vaccine was determined by coomassie brilliant blue staining and western blotting, After 3 subcutaneous injections of E.coli LLO/OVA, the percentages of CD3^＋CD4^＋T, CD4^＋CD25^＋T, CD3^CD8^＋T and OVA257-264 SIINFEKL specific CD8^＋T cells were determined by flow cytomytry, and the tumor metastatic inhibition effect in B16 OVA melanoma challenged mice was observed. Results： Recombinant E.coli LLO/OVA was successfully constructed, and the expression of LLO and OVA of the vaccine was confirmed. After 3 subcutaneous injections of E.coli LLO/OVA and E.coli OVA in mice, the percentages of CD3^＋CD4^＋T, CD4^＋CD25^＋T and CD3^＋CD8^＋T cells were equivalent in the two groups of mice. However, there were significantly more OVA257-264 SIINFEKL specific CD8^＋T cells in E.coli LLO/OVA vaccinated mice than that in E.coli OVA vaccinated mice. The prophylactic E.coli LLO/OVA vaccination effectively prevented the tumor metastasis to lungs in B16 OVA melanoma challenged mice. Depletion of CD8^＋T cells significantly impaired the tumor inhibition effect of the vaccine in B16 OVA challenged mice. The therapeutic vaccination of E.coli LLO/OVA significantly prevented melanoma metastasis to lungs in B I6 OVA challenged mice too. Conclusion： Recombinant E.coli LLO/OVA vaccination is highly effective in inhibiting murine malignant melanoma metastasis by promoting CD8^＋T cell immunity.
基金funded by Natural Science Foundation of Jiangsu Province (BK2007555)Science Innovation Engagement Fund of Yangzhou University (2008CXJ032)
文摘[ Objective] To explore different preservation methods of recombinant E. coli and find out the optimal conditions for preservation. [ Method] The recombinant E. coli DH5cx transformed pcDNA.3 were respectively preserved at 4℃ and -70 ℃, and the activity was determined after dif- ferent time. [ Result] The number of living E. coll with high dilutions preserved at 4 ℃ was gradually increased within the first 7 d, peaked on Day 7, and then gradually decreased. The number of living E. coli, which were preserved in 8% glycerol at -70℃ when OD800 at 0.8, were significantly higher than that of other groups after different preservation time. [ Conclusion] The optimal storage time was 7 d for recombinant E. coli at 4 ℃. For preservation at -70 ℃, the bacteria should be in logarithmic growth phase and preserved in 8% glycerol.
基金supported by National High-tech Research and Development Plan(863 Project)(2006AA10A207)
文摘The gene encoding the 18 kDa protein of Taenia solium metacestodes was amplified by RT-PCR and cloned into the pGEM-T vector for sequencing.The recombinant plasmid named pGEX-CE18 was constructed and transformed into E.coli BL21 for in vitro expression.SDS-PAGE and Western blot were employed for analyzing the recombinant protein,which was then used for development of an indirect ELISA for detection of anti-cysticercosis antibodies.The results showed that the recombinant protein of interest was 35 kDa in size,accounting for 28%of total bacteria proteins,and reacted with positive sera against cysticercosis.Using the newly-constructed indirect ELISA and a commercially available ELISA kit,paired analyses of 178 serum samples indicated that the concordant rate was 98.83%and the ELISA exhibited good specificity and sensitivity,supporting its utility and application for diagnosis of cysticercosis.
文摘The photosynthetic bacteria, Rodospirillum rubrum, produced hydrogen when grown in cheese whey in presence of light. The production increased three times as much when whey was used after being incubated in presence of Escherichia coli at 37℃ for 5 days, giving a total of 3600 ml of H2 in 10 days. The presence of Fe ions (5 mg/L) enhanced H2 production of treated whey to about 6000 ml in 10 days. Mo ions (0.3 and 1.6 mg/l) did not affect achieved H2 production of treated whey. However, when Fe and Mo ions were present together, the production was comparable with that of Mo ions alone, i.e. Mo prevented Fe of producing any enhancing effect. The addition of Mn ions (7.68 mg/L) to treated whey containing Fe (5 mg/L) and Mo ions (8 mg/L) increased H2 production to give about 9500 ml/10 days.
