A new biosensor platform was explored for detection of surfactant based on fluorescence changes from single strand DNA （ssDNA） and single-walled carbon nanotubes （SWNTs）. Thermodynamics assay was performed to valu...A new biosensor platform was explored for detection of surfactant based on fluorescence changes from single strand DNA （ssDNA） and single-walled carbon nanotubes （SWNTs）. Thermodynamics assay was performed to value the stability of probe. The affinities of SWNT to five common surfactants （SDS, DBS, Triton X-100, Tween-20 and Tween-80） were investigated by real-time fluorescence method. The effects of Mg^2＋ and pH on the fluorescence intensity of self-assembled quenched sensor were performed. The fluorescent emission spectra were used to measure the responses of self-assembled quenched fluorescent of ssDNA/SWNTs to different concentration surfactant（Triton X-100）. The FAM-DNA wrapped SWNTs probe was stable in a wide temperature range （5 ℃ to 80℃）. The binding strength of surfactants and single-stranded DNA （ssDNA） on SWNTs surfaces was shown as follows： Triton X-100〉DBS〉Tween-20〉Tween-80〉ssDNA〉SDS, and the optimized reaction conditions included pH 7.4 and 10 mmol/L Mg2＋. The fluorescence of FAM-ssDNA wrapped SWNTs was proportionally recovered as a result of adding different concentrations of Triton X- 100, which realizes the quantitative detection of Triton X- 100.展开更多
AIM： Passive immunotherapy using antibody against hepatitis B surface antigen （HBsAg） has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high af...AIM： Passive immunotherapy using antibody against hepatitis B surface antigen （HBsAg） has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal （5S） against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody.METHODS： We cloned the VH and V, genes of this mouse antibody, and fused them with CH1 domain of human IgG1 and C, domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. coli.RESULTS： The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. This chimeric antibody fragment was further expressed in different strains of E. coli to increase the yield.CONCLUSION： We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a fulllength chimeric antibody for therapeutic uses.展开更多
Design and development of efficient non-viral gene delivery systems is critical to overcome various barriers for effective intracellular gene delivery.Eight new spermine-based protonatable surfactants were designed,sy...Design and development of efficient non-viral gene delivery systems is critical to overcome various barriers for effective intracellular gene delivery.Eight new spermine-based protonatable surfactants were designed,synthesized and evaluated as non-viral pH-sensitive gene carriers.These carriers formed stable complexes with plasmid DNA at an N/P ratio as low as 2.The sizes of the carrier/pDNA nanoparticles (N/P = 12) were in the range of 90–130 nm,smaller than that of Lipofectamine2000/pDNA nanoparticles.The pDNA nanoparticles of the carriers exhibited substantially increased hemolysis when pH decreased from 7.4 to 5.5.The DNA nanoparticles had low cytotocixity,similar to that of Lipofectamine-2000/pDNA nanoparticles.The pH-sensitive gene carriers with no helper lipids resulted in much higher intracellular transfection and gene expression in U87 cells than Lipofectamine-2000.[N,N′-Bis(oleoylcysteinyl)(β-alanyl-α-lysyl)]-spermine monoamide (SKACO) resulted in the highest luciferase transfection efficiency,more than 400 times higher than that of Lipofectamine-2000,and GFP expression in 71% of transfected U87 cells.SKACO was identified as a promising lead carrier for efficient gene delivery.These spermine-based pH-sensitive amphiphilic carriers have a potential to be further developed as efficient non-viral multifunctional gene delivery systems.展开更多
基金Projects (21075032, 21005026, 21135001) supported by the National Natural Science Foundation of ChinaProject (llJJ5012) supported by Hunan Provincial Natural Science Foundation, China
文摘A new biosensor platform was explored for detection of surfactant based on fluorescence changes from single strand DNA （ssDNA） and single-walled carbon nanotubes （SWNTs）. Thermodynamics assay was performed to value the stability of probe. The affinities of SWNT to five common surfactants （SDS, DBS, Triton X-100, Tween-20 and Tween-80） were investigated by real-time fluorescence method. The effects of Mg^2＋ and pH on the fluorescence intensity of self-assembled quenched sensor were performed. The fluorescent emission spectra were used to measure the responses of self-assembled quenched fluorescent of ssDNA/SWNTs to different concentration surfactant（Triton X-100）. The FAM-DNA wrapped SWNTs probe was stable in a wide temperature range （5 ℃ to 80℃）. The binding strength of surfactants and single-stranded DNA （ssDNA） on SWNTs surfaces was shown as follows： Triton X-100〉DBS〉Tween-20〉Tween-80〉ssDNA〉SDS, and the optimized reaction conditions included pH 7.4 and 10 mmol/L Mg2＋. The fluorescence of FAM-ssDNA wrapped SWNTs was proportionally recovered as a result of adding different concentrations of Triton X- 100, which realizes the quantitative detection of Triton X- 100.
基金Supported by the Department of Biotechnology (DBT), Govt. of India, NO. BT/PR2540/PID/25/101/2001
文摘AIM： Passive immunotherapy using antibody against hepatitis B surface antigen （HBsAg） has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal （5S） against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody.METHODS： We cloned the VH and V, genes of this mouse antibody, and fused them with CH1 domain of human IgG1 and C, domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. coli.RESULTS： The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. This chimeric antibody fragment was further expressed in different strains of E. coli to increase the yield.CONCLUSION： We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a fulllength chimeric antibody for therapeutic uses.
文摘Design and development of efficient non-viral gene delivery systems is critical to overcome various barriers for effective intracellular gene delivery.Eight new spermine-based protonatable surfactants were designed,synthesized and evaluated as non-viral pH-sensitive gene carriers.These carriers formed stable complexes with plasmid DNA at an N/P ratio as low as 2.The sizes of the carrier/pDNA nanoparticles (N/P = 12) were in the range of 90–130 nm,smaller than that of Lipofectamine2000/pDNA nanoparticles.The pDNA nanoparticles of the carriers exhibited substantially increased hemolysis when pH decreased from 7.4 to 5.5.The DNA nanoparticles had low cytotocixity,similar to that of Lipofectamine-2000/pDNA nanoparticles.The pH-sensitive gene carriers with no helper lipids resulted in much higher intracellular transfection and gene expression in U87 cells than Lipofectamine-2000.[N,N′-Bis(oleoylcysteinyl)(β-alanyl-α-lysyl)]-spermine monoamide (SKACO) resulted in the highest luciferase transfection efficiency,more than 400 times higher than that of Lipofectamine-2000,and GFP expression in 71% of transfected U87 cells.SKACO was identified as a promising lead carrier for efficient gene delivery.These spermine-based pH-sensitive amphiphilic carriers have a potential to be further developed as efficient non-viral multifunctional gene delivery systems.
