title: Preparation and identification of scFv and bsFv against transferrin receptor.
abstract: 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.
authors.affiliation: Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China. liujing_81@126.com
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Daiwen
authors.initials: D
authors.lastname: Xiao
authors.forename: Xiaoou
authors.initials: X
authors.lastname: Zhou
authors.forename: Xue
authors.initials: X
authors.lastname: Wen
authors.forename: Hong
authors.initials: H
authors.lastname: Dai
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Wang
authors.forename: Xin
authors.initials: X
authors.lastname: Shen
authors.forename: Wei
authors.initials: W
authors.lastname: Dai
authors.forename: Daofeng
authors.initials: D
authors.lastname: Yang
authors.forename: Guanxin
authors.initials: G
authors.lastname: Shen
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D056945:Hep G2 Cells; D006801:Humans; D020014:K562 Cells; D008969:Molecular Sequence Data; D011990:Receptors, Transferrin / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies