抗重组人bFGF单克隆抗体可变区基因克隆及单链抗体的构建、表达.pdfVIP

bFGF bFGF B2F3 variable region V bFGF (single chain antibody fragmentsscFv)HB2151 bFGF MAb7 RNA RT-PCR VH VL ( splice-overlap extension, SOE)PCR VH VL linker(Gly Ser) 4 3 bFGF scFv pCANTAB 5E E.coli HB2151 SDSELISA VH 375 125 VL 399 133 4 (FRs) 3 (CDRs) scFv 789 263 V -linker-V SDS H L scFv 27kD ELASA scFv bFGF bFGF bFGF bFGF bFGF 1 bFGF ABSTRACT Aim: cloning of the variable region genes from hybridoma against bFGF to construct and express the single chain antibody (scFv) E.coli HB2151. Methods: Total RNA was extracted from hybridoma cell line B F secreting MAb against 2 3 bFGF and the cDNA was amplified by retropolymerase chain Reaction (RT-PCR). The VL and VH were fused by a short peptide linker containing 15 amino acids (Gly Ser) using 4 3 splice-overlap extension PCR to construct the scFv gene. The sequences of the scFv were analyzed by Shanghai Sangon Biological Engineering Technology Services Co. Ltd and Ig Blast data base in Gendbank. The scFv gene was inserted into pCANTAB-5E vector and expressed in E.coli HB2151. Results: The VH gene contained 375 base pairs and encoded 125 amine acid residues. The VL gene contained 399 base pairs and encoded 133 amine acid residues. There were four FRs, three CDRs and two characteristic c