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
Mycoplasma hominis is a member of the genus mycoplasma and has only been isolated from humans. It is most frequently isolated from the urogenital tract in the absence of symptoms, but has been isolated from wounds, br...Mycoplasma hominis is a member of the genus mycoplasma and has only been isolated from humans. It is most frequently isolated from the urogenital tract in the absence of symptoms, but has been isolated from wounds, brain abscess, inflamed joints, blood and placenta from pregnancy with adverse outcomes (especially preterm birth and occasionally term stillbirth). Controversy surrounds whether this organism is a commensal or a pathogen;however, Mycoplasma hominis has been shown to induce preterm birth and foetal lung injury in an experimental primate model as a sole pathogen. These bacteria are known to exist as a parasitic infection, due to a number of missing synthetic and metabolism pathway enzymes from their minimal genome;therefore, the ability to adhere to host cells is important. Here we provide a review that clarifies the different nomenclature (variable adherence-associated antigen and P50) that has been used to investigate the major surface adhesin for this organism, as well as reported mechanisms responsible for turning off its expression. Variation in the structure of this protein can be used to separate strains into six categories, a method that we were able to use to distinguish and characterise 12 UK strains isolated from between 1983 and 2012. We propose that the Vaa should be used in further investigations to determine if commensal populations and those that are associated with disease utilise different forms of this adhesin, as this is under-studied and identification of pathogenic determinants is overdue for this organism.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.
文摘Mycoplasma hominis is a member of the genus mycoplasma and has only been isolated from humans. It is most frequently isolated from the urogenital tract in the absence of symptoms, but has been isolated from wounds, brain abscess, inflamed joints, blood and placenta from pregnancy with adverse outcomes (especially preterm birth and occasionally term stillbirth). Controversy surrounds whether this organism is a commensal or a pathogen;however, Mycoplasma hominis has been shown to induce preterm birth and foetal lung injury in an experimental primate model as a sole pathogen. These bacteria are known to exist as a parasitic infection, due to a number of missing synthetic and metabolism pathway enzymes from their minimal genome;therefore, the ability to adhere to host cells is important. Here we provide a review that clarifies the different nomenclature (variable adherence-associated antigen and P50) that has been used to investigate the major surface adhesin for this organism, as well as reported mechanisms responsible for turning off its expression. Variation in the structure of this protein can be used to separate strains into six categories, a method that we were able to use to distinguish and characterise 12 UK strains isolated from between 1983 and 2012. We propose that the Vaa should be used in further investigations to determine if commensal populations and those that are associated with disease utilise different forms of this adhesin, as this is under-studied and identification of pathogenic determinants is overdue for this organism.
