Reactive oxygen species（ROS） plays a key role in human heart diseases. Glutathione peroxidase（GPX） functions as an antioxidant as it catalyzes the reduction of hydroperoxide. In order to investigate the antioxidan...Reactive oxygen species（ROS） plays a key role in human heart diseases. Glutathione peroxidase（GPX） functions as an antioxidant as it catalyzes the reduction of hydroperoxide. In order to investigate the antioxidant effect of human selenium-containing single-chain Fv（Se-scFv-B3）, a new mimic of GPX, a model system of hydrogen peroxide（H202）-induced rat cardiac myocyte damage was established. The cardiac myocyte damage was characte- rized in terms of cell viability, lipid peroxidation, cell membrane integrity, and intracellular H202 level. The Se-scFv-B3 significantly reduced H2O2-induced cell damage as shown by the increase of cell viability, the decline of malondialdehyde（MDA） production, lactate dehydrogenase（LDH） release, and intracellular H2O2 level. So Se-scFv-B3 may have a great potential in the treatment of human heart diseases induced by ROS.展开更多
Summary: A three-dimensional (3D) graphic model of a single-chain Fv (scFv) which was derived from an anti-human placental acidic isoferritin (PAF) monoclonal antibody (MAb) was construct- ed by a homologous protein...Summary: A three-dimensional (3D) graphic model of a single-chain Fv (scFv) which was derived from an anti-human placental acidic isoferritin (PAF) monoclonal antibody (MAb) was construct- ed by a homologous protein-predicting computer algorithm on Silicon graphic computer station. The structure, surface static electricity and hydrophobicity of scFv were investigated. Computer graphic modelling indicated that all regions of scFv including the linker, variable regions of the heavy (VH) and light (VL) chains were suitable. The VH region and the VL region were involved in composing the 'hydrophobic pocket'. The linker was drifted away VH and VL regions. The complementarity determining regions (CDRs) of VH and VL regions surrounded the 'hydrophobic pocket'. This study provides a theory basis for improving antibody affinity, investigating antibody structure and analyzing the functions of VH and VL regions in antibody activity.展开更多
Objective: To Obtain purified genetic eogineering recombinant scFv-fusion protein with potentialities of clinical application. Methods: Mouse anti-hepatocellular carcinoma (HCC) single chain Fv fragment (mscFv25 ) was...Objective: To Obtain purified genetic eogineering recombinant scFv-fusion protein with potentialities of clinical application. Methods: Mouse anti-hepatocellular carcinoma (HCC) single chain Fv fragment (mscFv25 ) was fused to human TNFa gene, then mscFv25-TNFa was subcloned into prokaryotic GST fusion expression vector pGEX 4T-l, and ex-. pressed in the host E. colt induced by IPTG. Expressed proteins as inclusion bodies were solubilized, solubilized and purified by GST affinity chromatography. The cytotoxity of mscFv25-TNFa was evaluated on SMMC-7721 by MTT, and the targeting therapeutic value was revealed in nude mice bearing HCC xenografts. Results: The specificity and affinity of mscFv25-TNFa were not markedly reduced compared with its parental antibody HAb25 against SMMC-7721 antigen. In vitro target cell SMMC-7721 was insensitive to mscFv25-TNFa. In the mscFv25-TNFa had certain targeting cytotoxicity and caused complete tumor disappearance in 4 of 14 mice, and the side effects of TNFa were much weaker in mscFv25-TNFa group than in group. Conclusion: SMMC-7721 may belong to the TNF-resistant type. While in the trial, mscFv25-TNFa caused complete tumor disappearance in 4 of 14 mice, but no disappearance in TNFa group, suggesting that mscFv25-TNFa had certain tar geting cytotoxicity, and the cytotoxicity of TNFa depended on some other factors in the. It may damage vascular endothelial cell and lead to ischemic necrosis, or induce the tumor cell to apoptosis and some agents to play the the of actinmycin-D in vivo. The targeting of mscFv25 can diminish unspecific cytotoxicity of TNFa, thus attenuate the side effects of TNFa.展开更多
Objective To obtain the gene of murine Single chain Fv fragment (ScFv) against human cervical cancer and to express it in E.coli . Methods The variable region gene fragments of the heavy and light chains, which were a...Objective To obtain the gene of murine Single chain Fv fragment (ScFv) against human cervical cancer and to express it in E.coli . Methods The variable region gene fragments of the heavy and light chains, which were amplified respectively using recombinant DNA techniques from CsA125 hybridoma cells, were spliced together through a flexible linker to ScFv against human cervical cancer. The ScFv genes were then cloned into expression vector pCANTAB 5E and expressed in E.coli HB2151 and TG1 respectively. The soluble ScFv were characterized by SDS-PAGE and Western blot. The antigen-binding activities of the soluble and phage displayed ScFv were assayed by ELISA and cell immunohistochemical analysis. Results The expressed ScFv antibodies were soluble and phage displayed. The soluble ScFv secreted and expressed in E.coli HB2151 induced by IPTG were confirmed with SDS-PAGE, Western blot and ELISA. The specific binding capacity of the soluble and phage displayed ScFv to the surface associated antigen of human cervical cancer cell line was further confirmed with immunohistochemical studies. Conclusion The soluble and phage displayed ScFv expressed in E.coli against human cervical cancer showed high, specific affinity for the cervical cancer cell line surface associated antigen.展开更多
In order to enhance the glutathione peroxidase（GPX） catalytic activity of the selenium-containing single-chain variable fragments（Se-scFv）, a novel human scFv was designed on the basis of the structure of human an...In order to enhance the glutathione peroxidase（GPX） catalytic activity of the selenium-containing single-chain variable fragments（Se-scFv）, a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional（3D） model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b（＋）, then the scFv protein was expressed in soluble form in Escherichia coli BL21（DE3） and purified by Ni2＋-immobilized metal affinity chromatography（IMAC）. The serine residue of scFv in the active site was converted into selenocysteine（Sec） with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mechanism.展开更多
基金Supported by the Grants from Department of Science and Technology of Jilin Province, China(No.20070726)Bureau of Science and Technology of Changchun City, China(No.2005038).
文摘Reactive oxygen species（ROS） plays a key role in human heart diseases. Glutathione peroxidase（GPX） functions as an antioxidant as it catalyzes the reduction of hydroperoxide. In order to investigate the antioxidant effect of human selenium-containing single-chain Fv（Se-scFv-B3）, a new mimic of GPX, a model system of hydrogen peroxide（H202）-induced rat cardiac myocyte damage was established. The cardiac myocyte damage was characte- rized in terms of cell viability, lipid peroxidation, cell membrane integrity, and intracellular H202 level. The Se-scFv-B3 significantly reduced H2O2-induced cell damage as shown by the increase of cell viability, the decline of malondialdehyde（MDA） production, lactate dehydrogenase（LDH） release, and intracellular H2O2 level. So Se-scFv-B3 may have a great potential in the treatment of human heart diseases induced by ROS.
文摘Summary: A three-dimensional (3D) graphic model of a single-chain Fv (scFv) which was derived from an anti-human placental acidic isoferritin (PAF) monoclonal antibody (MAb) was construct- ed by a homologous protein-predicting computer algorithm on Silicon graphic computer station. The structure, surface static electricity and hydrophobicity of scFv were investigated. Computer graphic modelling indicated that all regions of scFv including the linker, variable regions of the heavy (VH) and light (VL) chains were suitable. The VH region and the VL region were involved in composing the 'hydrophobic pocket'. The linker was drifted away VH and VL regions. The complementarity determining regions (CDRs) of VH and VL regions surrounded the 'hydrophobic pocket'. This study provides a theory basis for improving antibody affinity, investigating antibody structure and analyzing the functions of VH and VL regions in antibody activity.
文摘Objective: To Obtain purified genetic eogineering recombinant scFv-fusion protein with potentialities of clinical application. Methods: Mouse anti-hepatocellular carcinoma (HCC) single chain Fv fragment (mscFv25 ) was fused to human TNFa gene, then mscFv25-TNFa was subcloned into prokaryotic GST fusion expression vector pGEX 4T-l, and ex-. pressed in the host E. colt induced by IPTG. Expressed proteins as inclusion bodies were solubilized, solubilized and purified by GST affinity chromatography. The cytotoxity of mscFv25-TNFa was evaluated on SMMC-7721 by MTT, and the targeting therapeutic value was revealed in nude mice bearing HCC xenografts. Results: The specificity and affinity of mscFv25-TNFa were not markedly reduced compared with its parental antibody HAb25 against SMMC-7721 antigen. In vitro target cell SMMC-7721 was insensitive to mscFv25-TNFa. In the mscFv25-TNFa had certain targeting cytotoxicity and caused complete tumor disappearance in 4 of 14 mice, and the side effects of TNFa were much weaker in mscFv25-TNFa group than in group. Conclusion: SMMC-7721 may belong to the TNF-resistant type. While in the trial, mscFv25-TNFa caused complete tumor disappearance in 4 of 14 mice, but no disappearance in TNFa group, suggesting that mscFv25-TNFa had certain tar geting cytotoxicity, and the cytotoxicity of TNFa depended on some other factors in the. It may damage vascular endothelial cell and lead to ischemic necrosis, or induce the tumor cell to apoptosis and some agents to play the the of actinmycin-D in vivo. The targeting of mscFv25 can diminish unspecific cytotoxicity of TNFa, thus attenuate the side effects of TNFa.
文摘Objective To obtain the gene of murine Single chain Fv fragment (ScFv) against human cervical cancer and to express it in E.coli . Methods The variable region gene fragments of the heavy and light chains, which were amplified respectively using recombinant DNA techniques from CsA125 hybridoma cells, were spliced together through a flexible linker to ScFv against human cervical cancer. The ScFv genes were then cloned into expression vector pCANTAB 5E and expressed in E.coli HB2151 and TG1 respectively. The soluble ScFv were characterized by SDS-PAGE and Western blot. The antigen-binding activities of the soluble and phage displayed ScFv were assayed by ELISA and cell immunohistochemical analysis. Results The expressed ScFv antibodies were soluble and phage displayed. The soluble ScFv secreted and expressed in E.coli HB2151 induced by IPTG were confirmed with SDS-PAGE, Western blot and ELISA. The specific binding capacity of the soluble and phage displayed ScFv to the surface associated antigen of human cervical cancer cell line was further confirmed with immunohistochemical studies. Conclusion The soluble and phage displayed ScFv expressed in E.coli against human cervical cancer showed high, specific affinity for the cervical cancer cell line surface associated antigen.
基金Supported by the National Natural Science Foundation of China(No.30970608)the Applicative Technological Project of Bureau of Science and Technology of Changchun City, China(No.2009045)+1 种基金the Development and Planning Major Program of Jilin Provincial Science and Technology Department, China(No.20100948)the Innovation Method Fund of China (No.2008IM040800)
文摘In order to enhance the glutathione peroxidase（GPX） catalytic activity of the selenium-containing single-chain variable fragments（Se-scFv）, a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional（3D） model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b（＋）, then the scFv protein was expressed in soluble form in Escherichia coli BL21（DE3） and purified by Ni2＋-immobilized metal affinity chromatography（IMAC）. The serine residue of scFv in the active site was converted into selenocysteine（Sec） with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mechanism.
