To get recombinant antigen (Is/et Cell Autoantigen 69)ICA69 which was expressed in Escherichia coli strains (E.coli) by means of the gene engineering technique so that it can be used for early diagnosis of and screeni...To get recombinant antigen (Is/et Cell Autoantigen 69)ICA69 which was expressed in Escherichia coli strains (E.coli) by means of the gene engineering technique so that it can be used for early diagnosis of and screening in type Ⅰ diabetes mellitus, the cDNA fragment of human ICA69 was amplified by PCR, and then cloned into pSPORT 1 vector. After DNA sequencing, it was inserted into pGEX-2T between the sites of EcoR Ⅰ and Sma Ⅰ, then recombinant plasmid p2T-ICA69 was constructed and introduced into E.coli. The GST-ICA69 fusion protein was expressed by the induction of IPTG. The recombinant ICA69 proteins were used to detect the antibodies against hICA69 in 100 healthy subjects and type Ⅰ diabetic serum by the use of indirect ELISA. The sequence analysis showed that the amplified fragments contained 1449 bp, encoded 483 amino acids, and had been correctly inserted into pGEX-2T vector. The recombinant proteins expressed in the prokaryotic cells had immunogenicity and could be used to detect antibodies against ICA69 in type Ⅰ diabetic serum. Finally it can be concluded in this paper that the expression products obtained by the method of gene engineering are recombinant ICA69 antigen and may be used to improve the forecast rate and the diagnostic rate of type Ⅰ diabetes in combination with other tests.展开更多
AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase(AP). METHODS: The VH-linker-VL,namely scFv gene,was prepared by amplifying the VH and VL ...AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase(AP). METHODS: The VH-linker-VL,namely scFv gene,was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by 5/71 and Not I,it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1.The positive colonies were screened by colony PCR and their expressions were induced by IPTG.ScFv gene was gained by digesting ScFv expression vector pUC19/119 with 5/71 and NotI restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1.The positive colonies were selected by bacterial colony PCR.The expression of fusion protein (scFv-AP) was induced by IPTG.Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku.Immunofluorescent assay (IFA) demonstrated its reactivity with TfR.The molecular weight of scFv-AP was 75 ku.Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. CONCLUSION: We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP.It is promising for immunological experiments.展开更多
Abstract:Objective To investigate the protective effects of blocking CD40/CD40L interactions with human CD40-Ig fusion protein in a murine graft-versus-host disease model.Methods Human CD40 gene extracellular region w...Abstract:Objective To investigate the protective effects of blocking CD40/CD40L interactions with human CD40-Ig fusion protein in a murine graft-versus-host disease model.Methods Human CD40 gene extracellular region was inserted into plasmid pIG1, which contains genomic human IgG1 Fc gene. A transient vector containing CD40-Fc fusion gene was transfected into COS-7 cells. The CD40-Ig fusion protein was detected through enzyme-linked immunosorbent assay (ELISA). A constitutive vector was also generated by ligating the CD40-Fc fusion gene into pcDNA3.1 and transfecting it into CHO cells. CD40-Ig was purified by protein A affinity chromatography. SDS-PAGE, Western blot and ligand binding assay were used to identify the qualities of CD40-Ig. Murine acute graft-versus-host disease (GVHD) was induced by intravenous injection of C57BL/6J (H-2b) spleen cells into sub-lethally irradiated BALB/c (H-2d) mice. Protective effects against murine graft-versus-host disease by in vivo administration of CD40-Ig were evaluated.Results Mammalian expression vectors pIG/40Ig and p3.1/40Ig were constructed as described above. Chimeric proteins were expressed in COS-7 and CHO cell culture supernatant and confirmed by ELISA and Western blot. SDS-PAGE showed that fusion proteins had a disulfide-bonded dimeric structure and existed as homodimer. Purified CD40-Ig could bind to CD40L. In vivo administration of CD40-Ig could prevent the development of GVHD and significantly prolong the mean survival time of mice with graft-versus-host disease.Conclusions These results demonstrate that CD40/CD40L interactions play an important role in the pathogenesis of graft-versus-host disease and suggest clinical potential for CD40-Ig in the prevention and treatment of human graft-versus-host disease.展开更多
Antibody-based fusion proteins are the next generation of antibody therapies for cancer and other diseases.CD20 antigen,which is overexpressed on cell membranes in nearly 95% of cases of B-cell Non-Hodgkin's Lymph...Antibody-based fusion proteins are the next generation of antibody therapies for cancer and other diseases.CD20 antigen,which is overexpressed on cell membranes in nearly 95% of cases of B-cell Non-Hodgkin's Lymphoma,is an attractive target for the therapy of B-lymphoid malignancies.Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic that now has entered phase II clinical trials.In this study,we prepared an engineered fusion protein,scFv-LDP,consisting of an anti-CD20 scFv fragment and the apoprotein LDP of LDM using DNA recombination.After purification and refolding,scFv-LDP was found to bind specifically to CD20-positive lymphoma cells using ELISA and indirect immunofluorescent cytochemical staining assays.The energized fusion protein scFv-LDP-AE was obtained using molecular reconstitution of the active chromophore AE of LDM and scFv-LDP.MTT assay revealed potent cytotoxicity of scFv-LDP-AE to CD20-positive Raji and Daudi cells,with IC 50 values of 1.21×10-11 and 6.24×10-11 mol L-1,respectively.An in vivo subcutaneous xenograft model of CD20-positive B cell lymphoma in BALB/c (nu/nu) mice was also utilized.Drugs were given intravenously on day 14 and 21 after tumor transplantation.In terms of maximal tolerated doses,scFv-LDP-AE at 0.3 mg kg-1 suppressed tumor growth by 79.3%,and LDM at 0.05 mg kg-1 by 68.6% (P<0.05).Results suggested scFv-LDP-AE could be a potential candidate for tumor-targeting therapy.展开更多
文摘To get recombinant antigen (Is/et Cell Autoantigen 69)ICA69 which was expressed in Escherichia coli strains (E.coli) by means of the gene engineering technique so that it can be used for early diagnosis of and screening in type Ⅰ diabetes mellitus, the cDNA fragment of human ICA69 was amplified by PCR, and then cloned into pSPORT 1 vector. After DNA sequencing, it was inserted into pGEX-2T between the sites of EcoR Ⅰ and Sma Ⅰ, then recombinant plasmid p2T-ICA69 was constructed and introduced into E.coli. The GST-ICA69 fusion protein was expressed by the induction of IPTG. The recombinant ICA69 proteins were used to detect the antibodies against hICA69 in 100 healthy subjects and type Ⅰ diabetic serum by the use of indirect ELISA. The sequence analysis showed that the amplified fragments contained 1449 bp, encoded 483 amino acids, and had been correctly inserted into pGEX-2T vector. The recombinant proteins expressed in the prokaryotic cells had immunogenicity and could be used to detect antibodies against ICA69 in type Ⅰ diabetic serum. Finally it can be concluded in this paper that the expression products obtained by the method of gene engineering are recombinant ICA69 antigen and may be used to improve the forecast rate and the diagnostic rate of type Ⅰ diabetes in combination with other tests.
基金Supported by Natural Key and Basic Research Development Program,No.2002CB513109
文摘AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase(AP). METHODS: The VH-linker-VL,namely scFv gene,was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by 5/71 and Not I,it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1.The positive colonies were screened by colony PCR and their expressions were induced by IPTG.ScFv gene was gained by digesting ScFv expression vector pUC19/119 with 5/71 and NotI restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1.The positive colonies were selected by bacterial colony PCR.The expression of fusion protein (scFv-AP) was induced by IPTG.Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku.Immunofluorescent assay (IFA) demonstrated its reactivity with TfR.The molecular weight of scFv-AP was 75 ku.Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. CONCLUSION: We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP.It is promising for immunological experiments.
文摘Abstract:Objective To investigate the protective effects of blocking CD40/CD40L interactions with human CD40-Ig fusion protein in a murine graft-versus-host disease model.Methods Human CD40 gene extracellular region was inserted into plasmid pIG1, which contains genomic human IgG1 Fc gene. A transient vector containing CD40-Fc fusion gene was transfected into COS-7 cells. The CD40-Ig fusion protein was detected through enzyme-linked immunosorbent assay (ELISA). A constitutive vector was also generated by ligating the CD40-Fc fusion gene into pcDNA3.1 and transfecting it into CHO cells. CD40-Ig was purified by protein A affinity chromatography. SDS-PAGE, Western blot and ligand binding assay were used to identify the qualities of CD40-Ig. Murine acute graft-versus-host disease (GVHD) was induced by intravenous injection of C57BL/6J (H-2b) spleen cells into sub-lethally irradiated BALB/c (H-2d) mice. Protective effects against murine graft-versus-host disease by in vivo administration of CD40-Ig were evaluated.Results Mammalian expression vectors pIG/40Ig and p3.1/40Ig were constructed as described above. Chimeric proteins were expressed in COS-7 and CHO cell culture supernatant and confirmed by ELISA and Western blot. SDS-PAGE showed that fusion proteins had a disulfide-bonded dimeric structure and existed as homodimer. Purified CD40-Ig could bind to CD40L. In vivo administration of CD40-Ig could prevent the development of GVHD and significantly prolong the mean survival time of mice with graft-versus-host disease.Conclusions These results demonstrate that CD40/CD40L interactions play an important role in the pathogenesis of graft-versus-host disease and suggest clinical potential for CD40-Ig in the prevention and treatment of human graft-versus-host disease.
基金supported by the National High Technology Research and Development Program of China(Grant No. 2006AA02A255)the National Natural Science Foundation of China(Grant No. 30701029)the National Science and Technology Major Projects(Grant Nos.2009ZX09103-720 and 2010ZX09401-407)
文摘Antibody-based fusion proteins are the next generation of antibody therapies for cancer and other diseases.CD20 antigen,which is overexpressed on cell membranes in nearly 95% of cases of B-cell Non-Hodgkin's Lymphoma,is an attractive target for the therapy of B-lymphoid malignancies.Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic that now has entered phase II clinical trials.In this study,we prepared an engineered fusion protein,scFv-LDP,consisting of an anti-CD20 scFv fragment and the apoprotein LDP of LDM using DNA recombination.After purification and refolding,scFv-LDP was found to bind specifically to CD20-positive lymphoma cells using ELISA and indirect immunofluorescent cytochemical staining assays.The energized fusion protein scFv-LDP-AE was obtained using molecular reconstitution of the active chromophore AE of LDM and scFv-LDP.MTT assay revealed potent cytotoxicity of scFv-LDP-AE to CD20-positive Raji and Daudi cells,with IC 50 values of 1.21×10-11 and 6.24×10-11 mol L-1,respectively.An in vivo subcutaneous xenograft model of CD20-positive B cell lymphoma in BALB/c (nu/nu) mice was also utilized.Drugs were given intravenously on day 14 and 21 after tumor transplantation.In terms of maximal tolerated doses,scFv-LDP-AE at 0.3 mg kg-1 suppressed tumor growth by 79.3%,and LDM at 0.05 mg kg-1 by 68.6% (P<0.05).Results suggested scFv-LDP-AE could be a potential candidate for tumor-targeting therapy.
