Anti-lung Cancer Single Chain Antibody Construct, Expression, Purification And Preliminary Identification Of Its Activity

With the development of molecular biology, the technologies of antibody changed increasingly from cell engineering antibody to the gene-engineering antibody. Especially, the research on the theory and application of single-chain antibody was paid more attention. A monoclonal antibody is secreted from LC-1,which single chain Fv fragment(ScFv) named LC-1 ScFv was constructed based on recombinant phage displayed techniques. ScFv is the smallest segment being able to interact with antigen Because of small molecular weight and lack of Fc,which compares conventional antigen with strong force of penetration and immunogenecity.The total RNA was solated from high secretory volume and activity antibody LC-1 monocolony cells, which was transferred cDNA. The variable regions of heavy chain and light chain were cloned through PCR according to ScFv gene, respectively . ScFv' gene was formed after the SOE ligation of VH and modified VL. The ScFv gene amplified by PCR is cloned into pET-22b(+) plasmid between the Nco I/Sal I. and introduced into E.coliBL21 the constructed plasmid gave a high level of expression after IPTG induction..SDS-PAGE analyzed the purity of protein through the purification of Ni-NTA resin after IPTG induction. ELISA indicated that ScFv has activity similar to natural antibody.Recombinant antibodies and their fragments now represent over 30% of all biological proteins undergoing clinical trials for diagnosis and therapy. The focus on antibodies as the ideal cancer-targeting reagents recengtly culminated in approval by the Food and Drugs Administration for the first engineering therapeutic antibodies. In one strategies for cancer diagnosis and therapy, recombinant antibody fragments have been fused to radioisotopes, drugs, toxins, enzymes and biosensor surfaces. Bispecific antibodies and related fusion proteins have been produced for cancer immunotherapy, effectively enhancing the human immune response in anticancer vaccines and T cell recruitment strategies.