Extrahepatic biliary obstruction promotes intestinal translocation of bacteria and endotoxin and this process is an important cause of morbidity and mortality in patients with jaundice. This study was undertaken to in...Extrahepatic biliary obstruction promotes intestinal translocation of bacteria and endotoxin and this process is an important cause of morbidity and mortality in patients with jaundice. This study was undertaken to investigate the effect and mechanism of recombinant human growth hormone (rhGH) and to alleviate intestinal translocation of bacteria and endotoxin in murine obstructive jaundice. METHODS:A group of 42 Wistar rats were divided into 3 groups:sham operation (SO), bile duct ligation (BDL), and BDL and rhGH treatment (rhGH). By the end of the experiment,on day 7, the animals were killed, and their liver function and serum endotoxin were measured, bacterial cultures of the liver, kidney and mesenchymal lymph were made. Terminal ileum mucosa was observed under an electron microscope. RESULTS:Liver function was improved more significantly in the rhGH group than in the BDL group. The value of endotoxin in the rhGH group was 0.38±0.03 EU/ml, significantly lower than that in the BDL group (0.65±0.04 EU/ml, P【0.01), and similar to that in the SO group (0.30±0.02 EU/ml, P】0.05). The rate of bacteria translocation in the liver, kidney and mesenteric lymph was much higher in the BDL group than in other two groups. The rate of bacteria translocation in mesenteric lymph was 64.29%,significantly higher than that in the SO group and the rhGH group (P【0.05). There was no significant difference in bacteria translocation rate between the SO group and the rhGH group (P】0.05). Under an electron microscope , ileum mucosa epithelial cells in the BDL group were necrotic, and organelle were markedly metamorphic. In the rhGH group, ultrastructural changes were less evident or similar to those in the SO group. CONCLUSION:rhGH has significant protective effects on intestinal mucosa barrier in obstructive jaundice, and reduces intestinal translocation of bacteria and endotoxin.展开更多
Objective To construct a rapid and high-throughput assay for identifying recombinant bacteria based on mass spectrometry. Methods Matrix assisted laser desorption ionization time-of-flight mass spectrometry （MALDI-TO...Objective To construct a rapid and high-throughput assay for identifying recombinant bacteria based on mass spectrometry. Methods Matrix assisted laser desorption ionization time-of-flight mass spectrometry （MALDI-TOF MS） techniques were used to identify 12 recombinant proteins （10 of Yersinia pestis, 1 of Campylobacter jejuni and 1 of Helicobacter pylori）. A classification model for the various phase of recombinant bacteria was established, optimized and validated, using MALDI-TOF MS-CIinProTools system. The differences in the peptide mass spectra were analyzed by using Biotyper and FIexAnalysis softwares. Results Models of GA, SNN, and QC were established. After optimizing the parameters, the GA recognition model showed good classification capabilities： RC=100%, mean CVA=98.7% （the CVA was 96.4% in phase 1, 100% in phase 2, 98.4% in phase 3, and 100% in phase 4, respectively） and PPV=95}. This model can be used to classify the bacteria and their recombinant, which only requires 3.7x103 cells for analysis. The total time needed is only 10 min from protein extraction to reporting the result for one sample. Furthermore, this assay can automatically detect and test 96 samples concurrently. A total of 48 specific peaks （9, 16, 9, and 14 for the four stages, respectively） was found in the various phase of recombinant bacteria. Conclusion MALDI-TOF MS can be used as a fast, accurate, and high-throughput method to identify recombinant bacteria, which provide a new ideas not only for recombinant bacteria but also for the identification of mutant strains and bioterrorism pathogens.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
According to the sequence of the bile salt hydrolase （BSH） gene of Bifidobacterium and the restriction enzyme cutting sites of expression vector pNZ8148, primers were designed and the bile salt hydrolase （BSH） gen...According to the sequence of the bile salt hydrolase （BSH） gene of Bifidobacterium and the restriction enzyme cutting sites of expression vector pNZ8148, primers were designed and the bile salt hydrolase （BSH） gene was gotten from Bacillus bifidus ATCC 29521 by PCR. BSH gene was inserted into lactic acid bacteria expression vector pNZ8148 to construct the recombinant pNZ8148-BSH. The recombinant pNZ8148-BSH was transferred into lactic acid bacteria NZ9000 with electrotransformation method. And the recombinant which could express BSH protein was obtained. It was identified by SDS-PAGE electrophoresis and activity verification. The result could provide a rationale reference for expressing BSH in lactic acid bacteria.展开更多
Mercury and its organic compounds have been of severe concern worldwide due to their damage to the ecosystem and human health. The development of effective and affordable technology to monitor and signal the presence ...Mercury and its organic compounds have been of severe concern worldwide due to their damage to the ecosystem and human health. The development of effective and affordable technology to monitor and signal the presence of bioavailable mercury is an urgent need.The Mer gene is a mercury-responsive resistant gene, and a mercury-sensing recombinant luminescent bacterium using the Mer gene was constructed in this study. The mer operon from marine Pseudomonas putida strain SP1 was amplified and fused with prompterless lux CDABE in the p UCD615 plasmid within Escherichia coli cells, resulting in p THE30–E. coli.The recombinant strain showed high sensitivity and specificity. The detection limit of Hg^2＋was 5 nmol/L, and distinct luminescence could be detected in 30 min. Cd^2＋, Cu^2＋, Zn^2＋, Ca^2＋,Pb^2＋, Mg^2＋, Mn^2＋, and Al^3＋did not interfere with the detection over a range of 10-5–1 m M.Application of recombinant luminescent bacteria testing in environmental samples has been a controversial issue： especially for metal-sensing recombinant strains, false negatives caused by high cytotoxicity are one of the most important issues when applying recombinant luminescent bacteria in biomonitoring of heavy metals. In this study, by establishing an internal standard approach, the false negative problem was overcome;furthermore, the method can also help to estimate the suspected mercury concentration,which ensures high detection sensitivity of bioavailable Hg2＋.展开更多
文摘Extrahepatic biliary obstruction promotes intestinal translocation of bacteria and endotoxin and this process is an important cause of morbidity and mortality in patients with jaundice. This study was undertaken to investigate the effect and mechanism of recombinant human growth hormone (rhGH) and to alleviate intestinal translocation of bacteria and endotoxin in murine obstructive jaundice. METHODS:A group of 42 Wistar rats were divided into 3 groups:sham operation (SO), bile duct ligation (BDL), and BDL and rhGH treatment (rhGH). By the end of the experiment,on day 7, the animals were killed, and their liver function and serum endotoxin were measured, bacterial cultures of the liver, kidney and mesenchymal lymph were made. Terminal ileum mucosa was observed under an electron microscope. RESULTS:Liver function was improved more significantly in the rhGH group than in the BDL group. The value of endotoxin in the rhGH group was 0.38±0.03 EU/ml, significantly lower than that in the BDL group (0.65±0.04 EU/ml, P【0.01), and similar to that in the SO group (0.30±0.02 EU/ml, P】0.05). The rate of bacteria translocation in the liver, kidney and mesenteric lymph was much higher in the BDL group than in other two groups. The rate of bacteria translocation in mesenteric lymph was 64.29%,significantly higher than that in the SO group and the rhGH group (P【0.05). There was no significant difference in bacteria translocation rate between the SO group and the rhGH group (P】0.05). Under an electron microscope , ileum mucosa epithelial cells in the BDL group were necrotic, and organelle were markedly metamorphic. In the rhGH group, ultrastructural changes were less evident or similar to those in the SO group. CONCLUSION:rhGH has significant protective effects on intestinal mucosa barrier in obstructive jaundice, and reduces intestinal translocation of bacteria and endotoxin.
基金supported by the National Key Program for Infectious Disease of China(Contract No.2013ZX10004216-002)Key Projects in the National Science&Technology Pillar Program during the 12th Five-year Plan Period(Contract No.2012BAI06B02)the Science and Technology Program of Zhejiang Province Public Technology Social Development Project(No.2010C33035)
文摘Objective To construct a rapid and high-throughput assay for identifying recombinant bacteria based on mass spectrometry. Methods Matrix assisted laser desorption ionization time-of-flight mass spectrometry （MALDI-TOF MS） techniques were used to identify 12 recombinant proteins （10 of Yersinia pestis, 1 of Campylobacter jejuni and 1 of Helicobacter pylori）. A classification model for the various phase of recombinant bacteria was established, optimized and validated, using MALDI-TOF MS-CIinProTools system. The differences in the peptide mass spectra were analyzed by using Biotyper and FIexAnalysis softwares. Results Models of GA, SNN, and QC were established. After optimizing the parameters, the GA recognition model showed good classification capabilities： RC=100%, mean CVA=98.7% （the CVA was 96.4% in phase 1, 100% in phase 2, 98.4% in phase 3, and 100% in phase 4, respectively） and PPV=95}. This model can be used to classify the bacteria and their recombinant, which only requires 3.7x103 cells for analysis. The total time needed is only 10 min from protein extraction to reporting the result for one sample. Furthermore, this assay can automatically detect and test 96 samples concurrently. A total of 48 specific peaks （9, 16, 9, and 14 for the four stages, respectively） was found in the various phase of recombinant bacteria. Conclusion MALDI-TOF MS can be used as a fast, accurate, and high-throughput method to identify recombinant bacteria, which provide a new ideas not only for recombinant bacteria but also for the identification of mutant strains and bioterrorism pathogens.
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 863 Projects (2008AA10Z311)National Science and Technology Support Projects (2009BADB9B06)+1 种基金Started Post-doctoral Research Grant of Heilongjiang Province (LBH-Q07023)Harbin Technological Innovation of Special Funds (2007RFQXN020)
文摘According to the sequence of the bile salt hydrolase （BSH） gene of Bifidobacterium and the restriction enzyme cutting sites of expression vector pNZ8148, primers were designed and the bile salt hydrolase （BSH） gene was gotten from Bacillus bifidus ATCC 29521 by PCR. BSH gene was inserted into lactic acid bacteria expression vector pNZ8148 to construct the recombinant pNZ8148-BSH. The recombinant pNZ8148-BSH was transferred into lactic acid bacteria NZ9000 with electrotransformation method. And the recombinant which could express BSH protein was obtained. It was identified by SDS-PAGE electrophoresis and activity verification. The result could provide a rationale reference for expressing BSH in lactic acid bacteria.
基金supported by the National Natural Science Foundation of China (No. 21377065)the 863 National High-Tech Research and Development Program (No. 2014AA06A506)
文摘Mercury and its organic compounds have been of severe concern worldwide due to their damage to the ecosystem and human health. The development of effective and affordable technology to monitor and signal the presence of bioavailable mercury is an urgent need.The Mer gene is a mercury-responsive resistant gene, and a mercury-sensing recombinant luminescent bacterium using the Mer gene was constructed in this study. The mer operon from marine Pseudomonas putida strain SP1 was amplified and fused with prompterless lux CDABE in the p UCD615 plasmid within Escherichia coli cells, resulting in p THE30–E. coli.The recombinant strain showed high sensitivity and specificity. The detection limit of Hg^2＋was 5 nmol/L, and distinct luminescence could be detected in 30 min. Cd^2＋, Cu^2＋, Zn^2＋, Ca^2＋,Pb^2＋, Mg^2＋, Mn^2＋, and Al^3＋did not interfere with the detection over a range of 10-5–1 m M.Application of recombinant luminescent bacteria testing in environmental samples has been a controversial issue： especially for metal-sensing recombinant strains, false negatives caused by high cytotoxicity are one of the most important issues when applying recombinant luminescent bacteria in biomonitoring of heavy metals. In this study, by establishing an internal standard approach, the false negative problem was overcome;furthermore, the method can also help to estimate the suspected mercury concentration,which ensures high detection sensitivity of bioavailable Hg2＋.
