AIM:To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. METHODS: A large human naive scFv phage library was used to search f...AIM:To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. METHODS: A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DMA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in F.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. RESULTS: Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M, value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. CONCLUSION: The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.展开更多
The main approach to reduce graft rejection has been focused on the development of immunosuppressive agents at present.Although these strategies have reportedly reduced graft rejection,there has been a reciprocal incr...The main approach to reduce graft rejection has been focused on the development of immunosuppressive agents at present.Although these strategies have reportedly reduced graft rejection,there has been a reciprocal increase in more severe immunosuppression and lethal infections,as well as severe side effects.Blockade of costimulatory T cell response has been proved as one of useful strategies to reduce graft rejection.Furthermore, it has been shown that infusion of dendritic cells(DCs)with a potent negative regulatory ability for T cells could prolong allograft survival.In this study mouse DCs(mDCs)were transfected with the recombinant plasmid pcDNA3.0 containing mouse inducible costimulator-Ig(mICOS-Ig) cDNA by electroporation.The transient expression of mICOS-Ig in mDC could be detected by ELISA and SDS-PAGE.Mouse ICOS-Ig fusion protein expressed in mDC and mICOS-Ig gene-modified mDC could inhibit lymphocyte proliferation in mixed lymphocyte culture(MLC)in vitro.Furthermore,mICOS-Ig gene-modified mDC could inhibit lymphocyte proliferation in recipient mice.These results suggested that mICOS-Ig gene-modified mDC exerted inhibitory effects on T cells,and might be suitable for treatment or prevention of graft rejection and immunopathologic diseases.Cellular & Molecular Immunology.2004;1(2):153-157.展开更多
基金Supported by the Major State Basic Research Development Program of China, 973 Program, No. 2002CB513100
文摘AIM:To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. METHODS: A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DMA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in F.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. RESULTS: Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M, value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. CONCLUSION: The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.
基金surpported by National Key Basic Research Program of China(No.CB510008)
文摘The main approach to reduce graft rejection has been focused on the development of immunosuppressive agents at present.Although these strategies have reportedly reduced graft rejection,there has been a reciprocal increase in more severe immunosuppression and lethal infections,as well as severe side effects.Blockade of costimulatory T cell response has been proved as one of useful strategies to reduce graft rejection.Furthermore, it has been shown that infusion of dendritic cells(DCs)with a potent negative regulatory ability for T cells could prolong allograft survival.In this study mouse DCs(mDCs)were transfected with the recombinant plasmid pcDNA3.0 containing mouse inducible costimulator-Ig(mICOS-Ig) cDNA by electroporation.The transient expression of mICOS-Ig in mDC could be detected by ELISA and SDS-PAGE.Mouse ICOS-Ig fusion protein expressed in mDC and mICOS-Ig gene-modified mDC could inhibit lymphocyte proliferation in mixed lymphocyte culture(MLC)in vitro.Furthermore,mICOS-Ig gene-modified mDC could inhibit lymphocyte proliferation in recipient mice.These results suggested that mICOS-Ig gene-modified mDC exerted inhibitory effects on T cells,and might be suitable for treatment or prevention of graft rejection and immunopathologic diseases.Cellular & Molecular Immunology.2004;1(2):153-157.
