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
Transplant rejection,like tolerance,is a T cell-dependent event.There is compelling evidence to suggest that induction of transplant tolerance is an actively learned process in which T cells need to engage with the al...Transplant rejection,like tolerance,is a T cell-dependent event.There is compelling evidence to suggest that induction of transplant tolerance is an actively learned process in which T cells need to engage with the alloantigens in order to learn to tolerate the ailograft.A family of cytokines whose receptors use the same IL-2 receptor γc chain(also called the common γc)plays an important role in regulating multiple aspects of the aliograft response(i.e.rejection vs.tolerance).It is undeniable that γc cytokines can drive clonal expansion and effector maturation of alloreactive T cells,and therefore,targeting such cytokines or their receptor components remains an attractive way of blocking transplant rejection.However,we just started to appreciate that γc cytokines also regulate the acquisition of transplant tolerance via programming activated T cells for apoptotic cell death and via guiding the evolution of regulatory T cells.Thus,understanding precisely the role of γc cytokines in regulating T cell homeostasis and T cell regulation is critically important in the induction of transplant tolerance.Cellular & Molecular Immunology.2004;1(3):167-172.展开更多
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Transplant rejection,like tolerance,is a T cell-dependent event.There is compelling evidence to suggest that induction of transplant tolerance is an actively learned process in which T cells need to engage with the alloantigens in order to learn to tolerate the ailograft.A family of cytokines whose receptors use the same IL-2 receptor γc chain(also called the common γc)plays an important role in regulating multiple aspects of the aliograft response(i.e.rejection vs.tolerance).It is undeniable that γc cytokines can drive clonal expansion and effector maturation of alloreactive T cells,and therefore,targeting such cytokines or their receptor components remains an attractive way of blocking transplant rejection.However,we just started to appreciate that γc cytokines also regulate the acquisition of transplant tolerance via programming activated T cells for apoptotic cell death and via guiding the evolution of regulatory T cells.Thus,understanding precisely the role of γc cytokines in regulating T cell homeostasis and T cell regulation is critically important in the induction of transplant tolerance.Cellular & Molecular Immunology.2004;1(3):167-172.
