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
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.
