| In recent years monoclonal antibodies(MAbs) have become accepted tools for the detection and treatment of breast cancer.A humanized anti-HER2 antibody (Herceptin) has been approved by the United States Federal Drag Administration for the treatment of breast cancer. The successes with antitumor antibodies in patients has led to renewed interest in the identification of novel tumor-associated antigens suitable for antibody targeting.Until recently, the production of antibodied was limited to very laborious and time-consuming processes involving animal immunization schemes and/or hybridoma generation. Phage display antibody library has now made it possible to clone antibody genes in bacteria. Such of antibody genes makes it technically feasible to produce antibodies quickly in bacterial cultures and to genetically manipulate their structure such that ultimately, antibodies to any antigen may be created. With phage display, tailor-made antibodies may be synthesized and selected to acquire the desired affinity of binding and specificity for in vitro and in vivo diagnosis, or for immunotherapy of human disease. One of the most successful approaches is to display single-chain Fv(scFv)antibodies on filamentous phage.scFv is an antigen-binding protein, composed of an immunoglobulin heavy-chain variable domain(VH) and a light-chain variable domain(VL) joined together by a flexible peptide linker. scFv is the smallest fragment that maintains the binding specificity and affinity of the entire antibody. Because of their small size, scFv fragment could be useful in tumor imaging and therapeutic strategies. Together with function-based selection procedure, antibody phage display technologies would open more challenging applications and provide a powerful tool for drug and target discovery. In this study, we constructed a phage display single-chain variable fragment(scFv) library against breast cancer cells and screen the specific antibodies against MCF-7 cells from the phage display library.The total RNA was isolated from spleens of BALB/C mice immunized with breast cancer cell line MCF-7. A set of degenerate oligonucleotide primers were designed and used to amplify VH and VL genes which were then joined into single chain variable fragment(scFv) by a DNA linker encoding peptide(Gly4Ser)3. The scFv fragments were cloned into a phagemid (pCANTAB5E) and expressed as a fusion protein with E tag and phage M13 p3 protein in E. coli TG1.The recombinant phage were rescued by being cocultured with helper phage M13KO7 .The scFv fusion protein was displayed on the surfaces of recombinant phages.Amplification of VH generated a DNA fragment with the expected length(about 340bp),while VL generated an expected 320bp fragment. The scFv DNA fragment is about 750bp.A repertoire of 1.2×106 clone phage display antibody library was constructed. The library was screened with MCF-7 cells by binding-elution-enrichment procedure.The positive recombinant phages were identified by ELISA with MCF-7 cells ,HepG2 cells and Hela cells. After five rounds of panning, a phage-scFv named phage scFv-507 binding MCF-7cells specifically was selected from the library.DNA sequence analysis showed scFv-507 DNA had 732bp and encoded 244 amino acid.The recombinant scFv-507 was transformed into nonsuppression E.coli Top10 that was allowed to express soluble scFv proteins by induction of IPTG. After induced by IPTG, the amber stop codon could be recognized and scFv-507-E tag fusion protein secreted into the periplasm of bacteria E.coli Top10. The E-tag at the C-terminus allowed the protein to be detected by Western blot. The relative molecular mass of expressed scFv-507 was about 32KD showed by SDS-PAGE and Western blot, which was consistent with expected molecular weight. To increase the yield of scFv-507in E.coli, the scFv-507 gene was transferred to an expression vector pET24a.The recombinant expression vector was transformed into E.coli BL21(DE3).After induced by IPTG,scFv-507 recombinant protein was expressed efficiently as inclusion bodi...
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