AIM: To construct and evaluate a polyvalent recombinant vaccine strain Shigella flexneri2a T32 against enterotoxigenic E.coli/(ETEC). METHODS: By using a host-plasmid balanced lethal system based on asd gene, a polyva...AIM: To construct and evaluate a polyvalent recombinant vaccine strain Shigella flexneri2a T32 against enterotoxigenic E.coli/(ETEC). METHODS: By using a host-plasmid balanced lethal system based on asd gene, a polyvalent recombinant strain was constructed to highly express CS3 and regularly express fusion enterotoxin of LIB subunit and mutant ST (LTB/STm) in a vaccine strain Shigella flexneri 2a T32 with specific deletion of asd gene. Fimbria CS3 was observed by immunofluorescence and electron microscopy assay. The security of LTB/STm was examined by ileal loop assay and suckling mouse assay. To evaluate this new candidate vaccine, it was compared with a previous vaccine strain in plasmid and protein level, growth assay and immunogenicity in Balb/c mice. RESULTS: The newly constructed vaccine expressed CS3 and grew better than the previously constructed vaccine except for the lower expression of LTB/STm. Serum IgG and mucosal IgA against CS3, LTB, ST, and host lipopolysaccharide (LPS) were produced after immunization of Balb/c mice by oral route with the new strain. The titers were not significantly different from the Balb/c mice with the previous strain. CONCLUSION: This novel candidate diarrheal vaccine can effectively induce serum and mucosal antibody responses against ETEC and Shigella.展开更多
AIM: To prepare a kind of magnetic iron-dextran nanopartides that was coated with anti-E.coli O157:H7 IgG, analyze its application conditions, and try to use it to isolate E.coli O157:H7 from foods. METHODS: Magnetic ...AIM: To prepare a kind of magnetic iron-dextran nanopartides that was coated with anti-E.coli O157:H7 IgG, analyze its application conditions, and try to use it to isolate E.coli O157:H7 from foods. METHODS: Magnetic iron-dextran nanopartides were prepared by the reaction of a mixture of ferric and ferrous ions with dextran polymers under alkaline conditions. The particles were coated with antiserum against E.coli O157: H7 by the periodate oxidation-borohydride reduction procedure. The oxidation time, amount of antibody coating the particles, amount of nanoparticles, incubation time and isolation time were varied to determine their effects on recovery of the organisms. Finally, the optimum conditions for isolating E.coli O157:H7 from food samples were established. RESULTS: E.coli O157:H7 can be isolated from samples within 15 min with the sensitivity of 101 CFU/mL or even less. In the presence of 108 CFU/mL of other organisms, the sensitivity is 101-102 CFU/mL. Nonspecific binding of other bacteria to the particles was not observed. Two and a half hours of enrichment is enough for the particles to detect the target from the food samples inoculated with 1 CFU/g. CONCLUSION: Isolation of target bacteria by immuno magnetic nanoparticles is an efficient method with high sensitivity and specificity. The technique is so simple that it can be operated in lab and field even by untrained personnel.展开更多
To investigate the expression of telomerase gene hTRT mRNA in HeLa cells and to obtain hTRT protein for futher study Methods. The gene for encoding hTRT catalytic domain was cloned b...To investigate the expression of telomerase gene hTRT mRNA in HeLa cells and to obtain hTRT protein for futher study Methods. The gene for encoding hTRT catalytic domain was cloned based on RT PCR amplification from HeLa cells and sequenced The cloned hTRTcDNA was in frame inserted into His tag fusion expression vector pEK318 The His tag hTRT fusion proteins were purified by Ni NTA chromatography and stained by western blotting Results. An approximately 620bp fragment was generated and cloned into pBluescript SK+between SalI and BamHI sites DNA sequencing showed the isolated fragment was consistent to those reported SDS PAGE present that a 17kDa protein was expressed stably in E coli JM109 harboring pEKTRT344 containing 6×His tag and hTRT 150aa, and the expression level of the protein was about 26% of the total bacterial proteins, while the expression of pEKTRT containing 6×His tag and hTRT 243aa was only detectable as 27 kDa band in western blotting Both of fusion proteins were purified by Ni NTA chromatography and showed single band(>95% purifity) in Coomassie Brilliant staining Western blotting confirmed that two proteins could be recognized by the Ni NTA AP conjugate Conclusions. The hTRT catalytic domain was highly conserved The expressed hTRT protein contained recognizable His tag, telomerase specific and strong antigenic epitops, which may be convenient for further investigation展开更多
Objective: To amplify human era (Hera) gene, then express it in E.coli. Methods: Human era gene, after amplified by PCR and identified by sequencing, was inserted into the expression vector pGEX-4T3 in which exogenous...Objective: To amplify human era (Hera) gene, then express it in E.coli. Methods: Human era gene, after amplified by PCR and identified by sequencing, was inserted into the expression vector pGEX-4T3 in which exogenous gene was controlled by Ptac promoter. The recombinant plasmid pGEX-Hera was transformed into DH5 (and induced with IPTG chemically. Results: The human era gene was amplified and the sequence was correct. When the bacteria with pGEX-Hera was induced, an anticipated 65 000 protein band appeared on SDS-PAGE gel and amounted to 23% of total bacterial protein. Conclusion: The human era gene has been successfully amplified and efficiently expressed in E.coli.展开更多
基金Supported by the National High Technology Research and Development Program of China (863 Program), No. 2001AA215211the Military Basic Research Foundation, No. 01Z026
文摘AIM: To construct and evaluate a polyvalent recombinant vaccine strain Shigella flexneri2a T32 against enterotoxigenic E.coli/(ETEC). METHODS: By using a host-plasmid balanced lethal system based on asd gene, a polyvalent recombinant strain was constructed to highly express CS3 and regularly express fusion enterotoxin of LIB subunit and mutant ST (LTB/STm) in a vaccine strain Shigella flexneri 2a T32 with specific deletion of asd gene. Fimbria CS3 was observed by immunofluorescence and electron microscopy assay. The security of LTB/STm was examined by ileal loop assay and suckling mouse assay. To evaluate this new candidate vaccine, it was compared with a previous vaccine strain in plasmid and protein level, growth assay and immunogenicity in Balb/c mice. RESULTS: The newly constructed vaccine expressed CS3 and grew better than the previously constructed vaccine except for the lower expression of LTB/STm. Serum IgG and mucosal IgA against CS3, LTB, ST, and host lipopolysaccharide (LPS) were produced after immunization of Balb/c mice by oral route with the new strain. The titers were not significantly different from the Balb/c mice with the previous strain. CONCLUSION: This novel candidate diarrheal vaccine can effectively induce serum and mucosal antibody responses against ETEC and Shigella.
基金Supported by the National High-technology Research and Development Program of China (863 Program), No. 2003AA302260
文摘AIM: To prepare a kind of magnetic iron-dextran nanopartides that was coated with anti-E.coli O157:H7 IgG, analyze its application conditions, and try to use it to isolate E.coli O157:H7 from foods. METHODS: Magnetic iron-dextran nanopartides were prepared by the reaction of a mixture of ferric and ferrous ions with dextran polymers under alkaline conditions. The particles were coated with antiserum against E.coli O157: H7 by the periodate oxidation-borohydride reduction procedure. The oxidation time, amount of antibody coating the particles, amount of nanoparticles, incubation time and isolation time were varied to determine their effects on recovery of the organisms. Finally, the optimum conditions for isolating E.coli O157:H7 from food samples were established. RESULTS: E.coli O157:H7 can be isolated from samples within 15 min with the sensitivity of 101 CFU/mL or even less. In the presence of 108 CFU/mL of other organisms, the sensitivity is 101-102 CFU/mL. Nonspecific binding of other bacteria to the particles was not observed. Two and a half hours of enrichment is enough for the particles to detect the target from the food samples inoculated with 1 CFU/g. CONCLUSION: Isolation of target bacteria by immuno magnetic nanoparticles is an efficient method with high sensitivity and specificity. The technique is so simple that it can be operated in lab and field even by untrained personnel.
文摘To investigate the expression of telomerase gene hTRT mRNA in HeLa cells and to obtain hTRT protein for futher study Methods. The gene for encoding hTRT catalytic domain was cloned based on RT PCR amplification from HeLa cells and sequenced The cloned hTRTcDNA was in frame inserted into His tag fusion expression vector pEK318 The His tag hTRT fusion proteins were purified by Ni NTA chromatography and stained by western blotting Results. An approximately 620bp fragment was generated and cloned into pBluescript SK+between SalI and BamHI sites DNA sequencing showed the isolated fragment was consistent to those reported SDS PAGE present that a 17kDa protein was expressed stably in E coli JM109 harboring pEKTRT344 containing 6×His tag and hTRT 150aa, and the expression level of the protein was about 26% of the total bacterial proteins, while the expression of pEKTRT containing 6×His tag and hTRT 243aa was only detectable as 27 kDa band in western blotting Both of fusion proteins were purified by Ni NTA chromatography and showed single band(>95% purifity) in Coomassie Brilliant staining Western blotting confirmed that two proteins could be recognized by the Ni NTA AP conjugate Conclusions. The hTRT catalytic domain was highly conserved The expressed hTRT protein contained recognizable His tag, telomerase specific and strong antigenic epitops, which may be convenient for further investigation
基金research grant from the National NaturalScience Foundation of China (No. 39870380, 39670006) the ScienceFoundation of PLA (
文摘Objective: To amplify human era (Hera) gene, then express it in E.coli. Methods: Human era gene, after amplified by PCR and identified by sequencing, was inserted into the expression vector pGEX-4T3 in which exogenous gene was controlled by Ptac promoter. The recombinant plasmid pGEX-Hera was transformed into DH5 (and induced with IPTG chemically. Results: The human era gene was amplified and the sequence was correct. When the bacteria with pGEX-Hera was induced, an anticipated 65 000 protein band appeared on SDS-PAGE gel and amounted to 23% of total bacterial protein. Conclusion: The human era gene has been successfully amplified and efficiently expressed in E.coli.
