Expression, Purification And Preliminary Identification Of Single Chain Fv Antibody Dimer Against Human Hepatocellular Carcinoma

Objective:Through the soluble expression and purification of single chain Fv antibody dimer(scFv-BDM2) against human hepatocellular carcinoma in E.coli,and preliminary identification its affinity and specificity,the next step for the construction of the ^99mTc marked scFvDM dimer and scFvDM dimer-fever magnetic nanoparticles that lay the foundation for the clinical application.Methods:1.The scFv-BDM2 would be cloned from E.coli TG1 and infected E.coli HB2151.After induced by IPTG,the soluble scFv-BDM2 were expressed in the bacteria periplasm of E.coli HB2151 abundantly.Using HiTrap Anti-E Tag column,The scFv-BDM2 were purified by affinity chromatography.2.Native polyacrylamide gel electrophoresis of purified scFv-BDM2 was analyzed.The concentration and bioactivity of purified scFv-BDM2 were measured by Bradford method and enzyme-linked immunosorbent assay(ELISA),the affinity constants of scFv-BDM2 was determinated by non-competitive enzyme immunoassay,and the specificity of scFv-BDM2 was analyzed by ELISA.Results:1.Native polyacrylamide gel electrophoresis analysis of purified scFv-BDM2 showed that a protein bands appeared,Molecular weights of purified scFv-BDM2 were about 60 kDa.The concentration of purified scFv-BDM2 was 16μg/ml.2.The bioactivity valence of purified scFv-BDM2 was 1:64,The affinity constants of the positive dimer clone was 3.735×10⁷mol/L^-1,and the primitive clone scFvDM was 2.44×10⁷mol/L^-1,The affinity constants of the positive clone scFv-BDM2 was1.53 fold higher than that of the primitive clone scFvDM.ELISA showed that the scFv-BDM2 had high specificity to hepatoma carcinoma cell,but not combined to normal hepatocyte,gastric cancer cells and bladder cancer cells.Conclusions:Single-chain Fv antibodies dimer for HCC was expressed by E.coli, with high purity,affinity and specificity were obtained after affinity chromatography.