Objective To explore the strategies which reduce the amount of xenoantigen Galα1, 3 Gal. Methods Human α-galactosidase gene and α1,2-fucosyltransferase gene were transferred into cul-tured porcine vascular endothel...Objective To explore the strategies which reduce the amount of xenoantigen Galα1, 3 Gal. Methods Human α-galactosidase gene and α1,2-fucosyltransferase gene were transferred into cul-tured porcine vascular endothelial cells PEDSV.15 and human α-galactosidase transgenic mice were produced. The Galα1,3Gal on the cell surface and susceptibility of cells to human antibody-mediated lysis were analyzed. Results Human α-galactosidase gene alone reduced 78% of Galα1,3Gal on PEDSV.15 cell surface while human α-galactosidase combined with α1,2-fucosyltransferase genes removed Galα1,3Gal completely. Decrease of Galα1,3Gal could reduce susceptibility of cells to human antibody-mediated lysis, especially during co-expression of α-galactosidase gene and α1,2-fucosyltransferase gene. RT-PCR indicated positive human α-galactosidase gene expression in all organs of positive human α-galacto-sidase transgenic F1 mice including heart, liver, kidney, lung, and spleen, the amount of Galα1,3Gal antigens on which was reduced largely. 58% of spleen cells from F1 mice were destroyed by comp-lement-mediated lysis compared with 24% of those from normal mice. Conclusions Human α-galactosidase gene and α1,2-fucosyltransferase gene effectively reduce the expression of Galα1,3Gal antigens on endothelial cell surface and confers resistance to human serum-mediated cytolysis. The expression of human α-galactosidase in mice can also eliminate the Galα1,3Gal antigens in most tissues and decrease the susceptibility of spleen cells to human serum-mediated cytolysis.展开更多
Our objective is to solve the lactose malabsorption and intolerance of human beings by combining micro-ecology path with genetic engineering technique. Plasmid pMG36e was used to clone and express a β-galactosidase g...Our objective is to solve the lactose malabsorption and intolerance of human beings by combining micro-ecology path with genetic engineering technique. Plasmid pMG36e was used to clone and express a β-galactosidase gene from L. delbrueckü bulgaricus strain 1.1480 in the Lactococcus lactis subsp. cremoris MG1363 and Lactococcus lactis subsp. lactis IL1403. The recombinant plasmid was preserved and proliferated in Escherichia coli ( E. coli) JM109, and transformed into MG1363 and IL1403 by electroporation. The protein expression was studied. ( 1 ) The bifidobacterium culture medium ( BBL) was suitable for the growth of the strain 1.1480. (2) With 13 amino acids at the N-terminus from the vector, β-gal- actosidase fusion protein (which retained the enzyme activity) could be successfully expressed in E. coli JM109, MG1363 and IL1403, but the expression quantity was larger in the former than in the latter two. (3) The SD sequence designed could be successfully recognized by both the E. coli and the Lactococcus lactis, but the expression level of the non-fusion β-galac- tosidase protein was lower than that of the fusion protein in the same host. The β-galactosidase genetically engineered E. coli JM109 is a useful tool to produce this enzyme in vitro . The signal peptide of the usp45 protein from the Lactococcus lac- tis can be added before the promoter sequence to promote β-galactosidase secretion from Lactococcus lactis . The potential ap- plication of the β-galactosidase genetically engineered MG1363 and IL1403 to cure the lactose malabsorption and lactose in- tolerance in both health food and medicine is promising.展开更多
An electrochemical assay was developed for β-D-galactosidase in coliform bacteria Escherichia coli (E. coli) using p-aminophenyl-β-D-galactopyranoside (PAPG) as substrate. The hydrolysis of PAPG by pure β-D-galact...An electrochemical assay was developed for β-D-galactosidase in coliform bacteria Escherichia coli (E. coli) using p-aminophenyl-β-D-galactopyranoside (PAPG) as substrate. The hydrolysis of PAPG by pure β-D-galactosidase was investigated based on the cyclic voltammetric (CV) behavior of p-aminophenyl (PAP). The results obtained using the pure enzyme showed that the addition of magnesium ion facilitated the enzyme-catalytic reaction. K m at the optimal magnesium ion concentration (5 mmol/L) was 0.90 mmol/L. The substrate was also used for coliformbacteria, where β-D-galactosidase in the bacteria cells was strongly induced by isopropyl-β-D-thiogalactopyranoside (IPTG). When the E. coli cell density exceeded 1.1×10 3 mL -1, the CV response was linearly related to the number of E. coli, the detection time was about one hour. This method can be developed into a portable biosensor for rapid assay of coliform bacteria.展开更多
Several features of adenoviruses make them anattractive option as a vehicle to transfer genes intoprimary malignant neoplasms in vivo. These viruseshave low pathogenicity in humans and are notneurotoxic. In addition, ...Several features of adenoviruses make them anattractive option as a vehicle to transfer genes intoprimary malignant neoplasms in vivo. These viruseshave low pathogenicity in humans and are notneurotoxic. In addition, high titers of the virus can beachieved to allow higher levels of gene transfectionefficiency than the other vector systems. In vivotumorigenicity of G422 glioblastoma cells transfectedwith IL-2 and/or IL-3 genes decreased significantly inour privious report. In this study, recombinantadenoviruses were used to evaluate the therapeuticpotential of combined IL-2/IL-3 gene therapy in thetreatment of established subcutaneous tumor model ofG422 glioblastoma. Murine IL-2, IL-3 recombinantadenoviruses (2×10~8 pfu) were injected directly展开更多
When the cellulase hydrolyses carboxymethyl-cellulose-Na(CMC-Na), it is not fit to formula, V=KE, between reaction velocity and concentration of enzyme. Therefore, a factor that inhibits this enzyme reaction, which is...When the cellulase hydrolyses carboxymethyl-cellulose-Na(CMC-Na), it is not fit to formula, V=KE, between reaction velocity and concentration of enzyme. Therefore, a factor that inhibits this enzyme reaction, which is called the cellulase endo-inhibitor, was discovered in the fermentative medium of Trichoderma spp. It was shown as a single band through electrophoresis, indicating it is homogeneous and the inhibitor展开更多
文摘Objective To explore the strategies which reduce the amount of xenoantigen Galα1, 3 Gal. Methods Human α-galactosidase gene and α1,2-fucosyltransferase gene were transferred into cul-tured porcine vascular endothelial cells PEDSV.15 and human α-galactosidase transgenic mice were produced. The Galα1,3Gal on the cell surface and susceptibility of cells to human antibody-mediated lysis were analyzed. Results Human α-galactosidase gene alone reduced 78% of Galα1,3Gal on PEDSV.15 cell surface while human α-galactosidase combined with α1,2-fucosyltransferase genes removed Galα1,3Gal completely. Decrease of Galα1,3Gal could reduce susceptibility of cells to human antibody-mediated lysis, especially during co-expression of α-galactosidase gene and α1,2-fucosyltransferase gene. RT-PCR indicated positive human α-galactosidase gene expression in all organs of positive human α-galacto-sidase transgenic F1 mice including heart, liver, kidney, lung, and spleen, the amount of Galα1,3Gal antigens on which was reduced largely. 58% of spleen cells from F1 mice were destroyed by comp-lement-mediated lysis compared with 24% of those from normal mice. Conclusions Human α-galactosidase gene and α1,2-fucosyltransferase gene effectively reduce the expression of Galα1,3Gal antigens on endothelial cell surface and confers resistance to human serum-mediated cytolysis. The expression of human α-galactosidase in mice can also eliminate the Galα1,3Gal antigens in most tissues and decrease the susceptibility of spleen cells to human serum-mediated cytolysis.
文摘Our objective is to solve the lactose malabsorption and intolerance of human beings by combining micro-ecology path with genetic engineering technique. Plasmid pMG36e was used to clone and express a β-galactosidase gene from L. delbrueckü bulgaricus strain 1.1480 in the Lactococcus lactis subsp. cremoris MG1363 and Lactococcus lactis subsp. lactis IL1403. The recombinant plasmid was preserved and proliferated in Escherichia coli ( E. coli) JM109, and transformed into MG1363 and IL1403 by electroporation. The protein expression was studied. ( 1 ) The bifidobacterium culture medium ( BBL) was suitable for the growth of the strain 1.1480. (2) With 13 amino acids at the N-terminus from the vector, β-gal- actosidase fusion protein (which retained the enzyme activity) could be successfully expressed in E. coli JM109, MG1363 and IL1403, but the expression quantity was larger in the former than in the latter two. (3) The SD sequence designed could be successfully recognized by both the E. coli and the Lactococcus lactis, but the expression level of the non-fusion β-galac- tosidase protein was lower than that of the fusion protein in the same host. The β-galactosidase genetically engineered E. coli JM109 is a useful tool to produce this enzyme in vitro . The signal peptide of the usp45 protein from the Lactococcus lac- tis can be added before the promoter sequence to promote β-galactosidase secretion from Lactococcus lactis . The potential ap- plication of the β-galactosidase genetically engineered MG1363 and IL1403 to cure the lactose malabsorption and lactose in- tolerance in both health food and medicine is promising.
文摘An electrochemical assay was developed for β-D-galactosidase in coliform bacteria Escherichia coli (E. coli) using p-aminophenyl-β-D-galactopyranoside (PAPG) as substrate. The hydrolysis of PAPG by pure β-D-galactosidase was investigated based on the cyclic voltammetric (CV) behavior of p-aminophenyl (PAP). The results obtained using the pure enzyme showed that the addition of magnesium ion facilitated the enzyme-catalytic reaction. K m at the optimal magnesium ion concentration (5 mmol/L) was 0.90 mmol/L. The substrate was also used for coliformbacteria, where β-D-galactosidase in the bacteria cells was strongly induced by isopropyl-β-D-thiogalactopyranoside (IPTG). When the E. coli cell density exceeded 1.1×10 3 mL -1, the CV response was linearly related to the number of E. coli, the detection time was about one hour. This method can be developed into a portable biosensor for rapid assay of coliform bacteria.
文摘Several features of adenoviruses make them anattractive option as a vehicle to transfer genes intoprimary malignant neoplasms in vivo. These viruseshave low pathogenicity in humans and are notneurotoxic. In addition, high titers of the virus can beachieved to allow higher levels of gene transfectionefficiency than the other vector systems. In vivotumorigenicity of G422 glioblastoma cells transfectedwith IL-2 and/or IL-3 genes decreased significantly inour privious report. In this study, recombinantadenoviruses were used to evaluate the therapeuticpotential of combined IL-2/IL-3 gene therapy in thetreatment of established subcutaneous tumor model ofG422 glioblastoma. Murine IL-2, IL-3 recombinantadenoviruses (2×10~8 pfu) were injected directly
文摘When the cellulase hydrolyses carboxymethyl-cellulose-Na(CMC-Na), it is not fit to formula, V=KE, between reaction velocity and concentration of enzyme. Therefore, a factor that inhibits this enzyme reaction, which is called the cellulase endo-inhibitor, was discovered in the fermentative medium of Trichoderma spp. It was shown as a single band through electrophoresis, indicating it is homogeneous and the inhibitor
