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Preparation and Identification of scFv and bsfv against Transferrin Receptor

Journal of Huazhong University of Science and Technology(Medical Sciences) 2008年第6期28卷 621-625页

作者：刘静肖代雯周小鸥文雪代红王志华沈昕代维杨道锋沈关心 Department of Immunology Tongji Medical CollegeHuazhong University of Science and Technology Clinical Laboratory Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital Department of Microbiology Jiujiang Medical College Department of Infectious Diseases Tongji HospitalTongji Medical CollegeHuazhong University of Science and Technology

To obtain single chain variable fragment （scFv） and bivalent single chain variable fragment （bsfv） against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy （VH） and FR4 of variable light （VL）, respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsfv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsfv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsfv respectively. Flow cytometry showed that scFv and bsfv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsfv was higher than that of the parent scFv.

关键词： gene sequence G4S linker scFv bsfv transferrin receptor

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