Aeromonas hydrophila (A. hydrophila) 4AK4 produced poly(3 hydroxybutyrate co 3 hydroxyhexanoate) (PHBHHx) with an almost constant 3 hydroxyhexanoate (3HHx) content of 10%15% from lauric acid and/or soybean oi...Aeromonas hydrophila (A. hydrophila) 4AK4 produced poly(3 hydroxybutyrate co 3 hydroxyhexanoate) (PHBHHx) with an almost constant 3 hydroxyhexanoate (3HHx) content of 10%15% from lauric acid and/or soybean oil. Both A. hydrophila 4AK4 and recombinant Escherichia coli (E. coli) JMU193 (pBH32) produced PHBHHx with controllable 3HHx content when fed lauric acid and another co substrate. With glucose or gluconate as the co substrate, the 3HHx content in the copolyester produced by A. hydrophila 4AK4 was reduced slightly from 12% to 9%. However, the 3HHx content in the copolyester produced by E. coli JMU193 (pBH32) was significantly reduced from 9% to 2% with fructose as the co substrate. These results show that regulation of 3HHx content in PHBHHx can be achieved using genetically engineered E. coli.展开更多
Objective To explore the immune stimulation effect of recombinant E.coli LLO/OVA on mice bone marrow-derived dendritic cells (BMDCs) and T lymphocytes in vitro.Methods After BMDCs stimulated by E.coli LLO/OVA,their ...Objective To explore the immune stimulation effect of recombinant E.coli LLO/OVA on mice bone marrow-derived dendritic cells (BMDCs) and T lymphocytes in vitro.Methods After BMDCs stimulated by E.coli LLO/OVA,their Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD) receptor signalling pathway were examined by superarray hybridization;and the priming effect of the vaccine activated BMDCs on CD4+T and CD8+T was determined by [3H]thymidine uptake and ELISA,the tumor cytotoxic effect of activated CD8+T cells was determined by cytotoxic assay.Results After BMDCs were activated by E.coli LLO/OVA via TLR4,NOD1 receptor and NF-κB signalling pathway,the expression of their surface molecules including MHC class Ⅰ,MHC class Ⅱ,CD40,CD80 and CD86 significantly up-regulated;the secretion of IL-12 and IFN-? increased also.The mature BMDCs stimulated the allergic CD4+T and CD8+T cells proliferation and their IL-2 and IFN-γ secretion,and the activated CD8+T cells effectively killed B16-OVA melanoma cells and RMA-S/OVA lymphoma cells in vitro.Conclusion E.coli LLO/OVA is effective in inducing BMDCs maturation via activating TLR4 and NOD1 receptor signalling pathway and promoting specific anti-tumor T cell immunity in vitro.展开更多
The ful length phytase gene of Mitsuokel a jalaludini was successful y cloned and was found to be 1 047 bp in length, with 348 amino acids, and was designated as PHY7 phytase gene. A comparison of the sequence of PHY7...The ful length phytase gene of Mitsuokel a jalaludini was successful y cloned and was found to be 1 047 bp in length, with 348 amino acids, and was designated as PHY7 phytase gene. A comparison of the sequence of PHY7 phytase gene of M. jalaludini with various microbial phytase gene sequences showed that it was not similar to those from other bacteria except Selenomonas ruminatium, thus suggesting that they may both express a new class of phytase. The PHY7 phytase gene was subsequently subcloned into bacterial expression vector, pET32a, for expression in Escherichia coli strain Ro-setta-gami. Expression of the recombinant phytase gene was optimised and characterised. The recombinant phytase was estimated to be approximately 55 kDa by SDS-PAGE analysis. The recombinant phytase exhibited optimum activity at 55°C, pH 4.5 and showed good pH stability from pH 3.5 to 5.5 (>78%relative activity). Metal ions such as Ca2+, Mg2+, and K+were found to exert signiifcant stimulatory effect on the recombinant phytase activity while Cu2+, Fe3+, and Zn2+greatly inhibited the enzyme activity. The recombinant phytase showed moderate resistance to trypsin proteolysis, but susceptible to pepsin proteolysis. The results of the study showed that several characteristics of recombinant phytase were slightly different from the native enzyme. Unfavourable characteristics such as reduced pH stability and metal ion effects should be taken into consideration during feed enzyme formulation.展开更多
The effect of temperature on the formation of recombinant protein,apolipoprotein A-IMilano was investigated in the present study.The temperature of the initial growth phase was set at 30ºC,while temperature varia...The effect of temperature on the formation of recombinant protein,apolipoprotein A-IMilano was investigated in the present study.The temperature of the initial growth phase was set at 30ºC,while temperature variation in induction phase was arranged in three modes.High cell-density culture of Escherichia coli and high expression of recombinant human by twice temperature-shifted induction were carried out.Experimental results showed that ApoA-IMilano reached 4.8 g/L with the final cell density of OD600,150.It was found that twice temperature-shifted induction could successfully avoid the effect of acetic acid on cell density and the expression of the product.The present study provides a basic procedure for the production of recombinant ApoA-IMilano.展开更多
A study of the effect of drug, kanamycin, on the growth metabolism of recombinant Escherichia coli B 1 was carded out by microcalorimeter monitoring of the metabolic activity of treated cells. Power-time curves of gro...A study of the effect of drug, kanamycin, on the growth metabolism of recombinant Escherichia coli B 1 was carded out by microcalorimeter monitoring of the metabolic activity of treated cells. Power-time curves of growing recombinant Escherichia coli cell suspensions, treated with different kanamycin doses, were recorded. The extent of the effect was evaluated by changes in the slopes of the microcalorimetric curves and the kinetics of the drug action was interpreted from the time at which these changes reached their maximum values and maintained their maximum values. Experimental dose-effect relationships conform to the median-effect principle of the mass-action law: fa/(1-fa)=(D/D50)^m. A plot of y=lg[(fa)^1-1]^-1 versus x=lg D gives the slope m, D50 and R∞. The experimental results revealed that high concentration of kanamycin had an inhibitory effect on the growth of recombinant Escherichia coli B 1 in the lg phase, and had a promoting effect in the stationary period. Moreover, it was demonstrated that microcalorimetry was a reliable method for the detection of modulatory effects in biology.展开更多
基金Supported by the National Natural Science Foundation of China (No. 2 0 0 74 0 2 0 ) and Tsinghua U niversity"985" Foundation
文摘Aeromonas hydrophila (A. hydrophila) 4AK4 produced poly(3 hydroxybutyrate co 3 hydroxyhexanoate) (PHBHHx) with an almost constant 3 hydroxyhexanoate (3HHx) content of 10%15% from lauric acid and/or soybean oil. Both A. hydrophila 4AK4 and recombinant Escherichia coli (E. coli) JMU193 (pBH32) produced PHBHHx with controllable 3HHx content when fed lauric acid and another co substrate. With glucose or gluconate as the co substrate, the 3HHx content in the copolyester produced by A. hydrophila 4AK4 was reduced slightly from 12% to 9%. However, the 3HHx content in the copolyester produced by E. coli JMU193 (pBH32) was significantly reduced from 9% to 2% with fructose as the co substrate. These results show that regulation of 3HHx content in PHBHHx can be achieved using genetically engineered E. coli.
基金supported by a grant from the State Scholarship Fund under the China Scholarship Council (No.2003850064)the Chongqing Education Commission (KJ080319)
文摘Objective To explore the immune stimulation effect of recombinant E.coli LLO/OVA on mice bone marrow-derived dendritic cells (BMDCs) and T lymphocytes in vitro.Methods After BMDCs stimulated by E.coli LLO/OVA,their Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD) receptor signalling pathway were examined by superarray hybridization;and the priming effect of the vaccine activated BMDCs on CD4+T and CD8+T was determined by [3H]thymidine uptake and ELISA,the tumor cytotoxic effect of activated CD8+T cells was determined by cytotoxic assay.Results After BMDCs were activated by E.coli LLO/OVA via TLR4,NOD1 receptor and NF-κB signalling pathway,the expression of their surface molecules including MHC class Ⅰ,MHC class Ⅱ,CD40,CD80 and CD86 significantly up-regulated;the secretion of IL-12 and IFN-? increased also.The mature BMDCs stimulated the allergic CD4+T and CD8+T cells proliferation and their IL-2 and IFN-γ secretion,and the activated CD8+T cells effectively killed B16-OVA melanoma cells and RMA-S/OVA lymphoma cells in vitro.Conclusion E.coli LLO/OVA is effective in inducing BMDCs maturation via activating TLR4 and NOD1 receptor signalling pathway and promoting specific anti-tumor T cell immunity in vitro.
基金The financial support provided by the Universiti Putra Malaysia under the Research University Grant Scheme and Ministry of Higher Education under the Long Term Research Grant Scheme, Malaysia
文摘The ful length phytase gene of Mitsuokel a jalaludini was successful y cloned and was found to be 1 047 bp in length, with 348 amino acids, and was designated as PHY7 phytase gene. A comparison of the sequence of PHY7 phytase gene of M. jalaludini with various microbial phytase gene sequences showed that it was not similar to those from other bacteria except Selenomonas ruminatium, thus suggesting that they may both express a new class of phytase. The PHY7 phytase gene was subsequently subcloned into bacterial expression vector, pET32a, for expression in Escherichia coli strain Ro-setta-gami. Expression of the recombinant phytase gene was optimised and characterised. The recombinant phytase was estimated to be approximately 55 kDa by SDS-PAGE analysis. The recombinant phytase exhibited optimum activity at 55°C, pH 4.5 and showed good pH stability from pH 3.5 to 5.5 (>78%relative activity). Metal ions such as Ca2+, Mg2+, and K+were found to exert signiifcant stimulatory effect on the recombinant phytase activity while Cu2+, Fe3+, and Zn2+greatly inhibited the enzyme activity. The recombinant phytase showed moderate resistance to trypsin proteolysis, but susceptible to pepsin proteolysis. The results of the study showed that several characteristics of recombinant phytase were slightly different from the native enzyme. Unfavourable characteristics such as reduced pH stability and metal ion effects should be taken into consideration during feed enzyme formulation.
基金This work was funded by the National Re-search Project for High-Tech Development(No.2002AA217021)supported by a Grant-aid for the Special Project of State Key Science and Technology.
文摘The effect of temperature on the formation of recombinant protein,apolipoprotein A-IMilano was investigated in the present study.The temperature of the initial growth phase was set at 30ºC,while temperature variation in induction phase was arranged in three modes.High cell-density culture of Escherichia coli and high expression of recombinant human by twice temperature-shifted induction were carried out.Experimental results showed that ApoA-IMilano reached 4.8 g/L with the final cell density of OD600,150.It was found that twice temperature-shifted induction could successfully avoid the effect of acetic acid on cell density and the expression of the product.The present study provides a basic procedure for the production of recombinant ApoA-IMilano.
基金Project supported by the National Science and Technology Key Funds (No. 2003DA901 A32) and the National Natural Science Foundation of China (No. 20476085).
文摘A study of the effect of drug, kanamycin, on the growth metabolism of recombinant Escherichia coli B 1 was carded out by microcalorimeter monitoring of the metabolic activity of treated cells. Power-time curves of growing recombinant Escherichia coli cell suspensions, treated with different kanamycin doses, were recorded. The extent of the effect was evaluated by changes in the slopes of the microcalorimetric curves and the kinetics of the drug action was interpreted from the time at which these changes reached their maximum values and maintained their maximum values. Experimental dose-effect relationships conform to the median-effect principle of the mass-action law: fa/(1-fa)=(D/D50)^m. A plot of y=lg[(fa)^1-1]^-1 versus x=lg D gives the slope m, D50 and R∞. The experimental results revealed that high concentration of kanamycin had an inhibitory effect on the growth of recombinant Escherichia coli B 1 in the lg phase, and had a promoting effect in the stationary period. Moreover, it was demonstrated that microcalorimetry was a reliable method for the detection of modulatory effects in biology.