Construction Of ScFv Library With Human Tumor From Immunised Mice And Screening Antibodies Binding To Breast Cancer

This work is the one part of the antibody library platform in our Lab. To facilitate our research in tumor molecular biology and investigate the diagnosis and therapy for tumor, we constructed a scFv library with human tumor cells from immunised mice and screened two antibodies binding to tumor cells.The recombinant phage antibody system was used to construct a single-chain Fv fragment (scFv) cDNA library. First BALB/c mice were immunised with human breast cancer cell lines MCF-7, T47D, MDA-MB-435, and total RNA was isolated from the spleens of immunized mice, RT-PCR was used to generate cDNA from total RNA. Then, heavy and light chain genes (VH and VL) of antibody were amplified separately and assembled into scFv gene with a specially constructed linker DNA by PCR, the scFv gene was ligated into the phagemid vector pCANTAB 5E and the ligated sample was transformed into competent E.coli TG1. A scFv library of 1.3 × 106 was obtained.After five rounds of panning with MCF-7 cells and normal human hepatocyte cell line HL02 as subtractive selection antigen, two phage-scFvs named scFv-873 and scFv-874 were selected with the ELISA from the enriched phages, which can bind to MCF-7 cells more strongly. The sequences of the two scFv genes were compared with the antibodies in Antibody Bank, and the results showed these two scFv genes were new genes.The phagemids containing two scFv gene fragments were transformed into the TOP10 strain, after scFvs were induced by 1PTG, the results of Western blot assay showed scFv-874 could be expressed in the supernate, the periplasm and whole cells of TOP10, but scFv-873 could be expressed in whole cells only. Then two scFv genes were inserted into pTIG-Trx vector, and scFvs could be expressed as soluble and functional form. The expressed antibodies were purified by Ni-NTA resin.