AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas.METHODS: mRNA was isolated from the hybridoma cell lineproducing MC3 and the DNA...AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas.METHODS: mRNA was isolated from the hybridoma cell lineproducing MC3 and the DNAs encoding variable domains ofheavy and light chains(VH and VL) oftthe antibody wereamplified separately byRT-PCR and assembled into ScFvDNA with a linker DNAThe ScFv DNA was iigated into thephagemid vector pCANTAB5E and the ligated sample wastransformed into E. coil TG1. The transformed cells wereinfected with M13KO7 helper phage to yield recombinantphages. After two rounds of panning with gastric carcinomacell line AGS highly expressing MC3-binding antigen, thephage clones displaying ScFv fragments of the antibodywere selected by ELISA. 4 phage clones showing strongsignal in ELISA were used to infect E. coil HB2151 toexpress soluble ScFvs. The soluble ScFve were identified byDot blot and Western blot, and their antigen-binding activitywas assayed by ELISA. The VH and VL DNAs of the ScFvDNA derived from phage clone 19 were sequenced.RESULTS: The VH, VL and ScFv DNAs were about 340 bp,320 bp and 750 bp respectively. After two rounds of panningto the recombinant phages, 18 antigen-positive phageclones were selected from 30 preselected phage clones byELISA. All the soluble ScFvs derived from the 4 out of the 18antigen-positive phage clones were about Mr 32 000 andconcentrated in periplasmatic space under the given culturecondition. The soluble ScFvs could bind the antigen, andthey shared the same binding site with MC3. The sequencesof the VH and VL DNAs of the MC3 ScFv showed that thevariable antibody genes belonged to the IgG1 subgroup,κ-type.CONCLUSION: The soluble ScFv of MC3 is successfullyproduced, which not only provides a possible novel targetingvehicle for in vivo and in vitro study on associated cancers,but also offers the anuibody a stable genetic source.展开更多
Single-chain variable domain fragment （scFv） 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pa...Single-chain variable domain fragment （scFv） 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pastoris. The afifnity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunolfuorescence staining. The ability of the fusion protein to block myas-thenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for speciifc immunosuppressive therapy of myasthenia gravis.展开更多
文摘AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas.METHODS: mRNA was isolated from the hybridoma cell lineproducing MC3 and the DNAs encoding variable domains ofheavy and light chains(VH and VL) oftthe antibody wereamplified separately byRT-PCR and assembled into ScFvDNA with a linker DNAThe ScFv DNA was iigated into thephagemid vector pCANTAB5E and the ligated sample wastransformed into E. coil TG1. The transformed cells wereinfected with M13KO7 helper phage to yield recombinantphages. After two rounds of panning with gastric carcinomacell line AGS highly expressing MC3-binding antigen, thephage clones displaying ScFv fragments of the antibodywere selected by ELISA. 4 phage clones showing strongsignal in ELISA were used to infect E. coil HB2151 toexpress soluble ScFvs. The soluble ScFve were identified byDot blot and Western blot, and their antigen-binding activitywas assayed by ELISA. The VH and VL DNAs of the ScFvDNA derived from phage clone 19 were sequenced.RESULTS: The VH, VL and ScFv DNAs were about 340 bp,320 bp and 750 bp respectively. After two rounds of panningto the recombinant phages, 18 antigen-positive phageclones were selected from 30 preselected phage clones byELISA. All the soluble ScFvs derived from the 4 out of the 18antigen-positive phage clones were about Mr 32 000 andconcentrated in periplasmatic space under the given culturecondition. The soluble ScFvs could bind the antigen, andthey shared the same binding site with MC3. The sequencesof the VH and VL DNAs of the MC3 ScFv showed that thevariable antibody genes belonged to the IgG1 subgroup,κ-type.CONCLUSION: The soluble ScFv of MC3 is successfullyproduced, which not only provides a possible novel targetingvehicle for in vivo and in vitro study on associated cancers,but also offers the anuibody a stable genetic source.
基金supported by the National Natural Science Foundation of China,No.30360100,30760234,30860260,81160373,81360458
文摘Single-chain variable domain fragment （scFv） 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pastoris. The afifnity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunolfuorescence staining. The ability of the fusion protein to block myas-thenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for speciifc immunosuppressive therapy of myasthenia gravis.
