[Construction of phage display antibody library to MCF-7 cells and screening of single-chain antibodies against breast cancer cells].
The aim of this study is to construct a phage display single-chain variable fragment (scFv) library against breast cancer cells and screen the specific antibodies against MCF-7 cells from the library. The BALB/C mice were immunized with MCF-7 cells. Total RNA of spleens was isolated. The heavy-chain (VH) and light-chain variable region genes (VL) of the antibodies were amplified by RT-PCR and joined into a single chain by overlapping PCR with a linker DNA encoding the peptide (Gly4Ser)3. The assembled scFv fragments were cloned into the phagemids(pCANTAB5E) and the recombinant phagemids were used to transform competent E. coli TG1. The transformed TG1 cells were infected by helper phage M13KO7 and the recombinant phagemids were rescued. The scFv fusion proteins were displayed on the surfaces of the recombinant phages. A phage display antibody library of repertoire of 1.2 x 10(6) clones was constructed. The specific antibodies against MCF-7 cells were enriched by 75 times after five rounds of affinity selection. Ten recombinant phages clones that exhibited specific binding to MCF-7 cells were identified. The specificity of those phage clones was analyzed by reactivity against HepG2 cells and Hela cells by ELISA. One of the selected phage clones against MCF-7cells was used to infect E. coli TOP10 to produce the soluble scFv antibodies after induction with IPTG. The strategy of construction and screening of antibody library directed against the whole tumor cells described in this report should be generally applicable to generate tumor cell-specific antibodies.
Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China.
Yan
Y
Zhao
Qing-Ming
QM
Wang
Xue-Qi
XQ
Fu
Ji-Zhong
JZ
Chen
Guo-Cai
GC
Fan
Hui-Peng
HP
Chen
D000818:Animals; D000912:Antibodies, Neoplasm / Q000235:genetics* / Q000276:immunology; D001943:Breast Neoplasms / Q000276:immunology* / Q000628:therapy; D045744:Cell Line, Tumor; D005260:Female; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D019151:Peptide Library; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology
D000912:Antibodies, Neoplasm; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D057127:Single-Chain Antibodies