Establishment Of Expression And Purification Protocol For E3 Human Single Chain Fv Antibody Against Amyloid β Protein And Identification Of Its Epitope

Amyloidα(Aβ) has been regarded as a primary causative factor of Alzheimer's disease. The abnormal polymerization and deposit of Aβplays a central role in pathogenesis of AD. Passive immunization of AD mouse models with antibodies against Aβprevented or reduced Aβdeposits and attenuated their memory and learning deficits. Most of these antibodies were derived from mice, which limited their application on human beings. Single chain variable fragment antibody from human scfv phage library seems more appropriate for AD clinical trial.Our group previously reported that we had obtained E3 scfv against Aβ_1-40 by screening human scfv phage library and identified some properties of this E3 scfv in vitro.In this study, we used IPTG in different concentration to induce expression of E3 scfv under fixed condition which inducing time was 18 hours and inducing temperature was 20℃and analyzed its production by SDS-PAGE assay. The result suggested that 0.1 mM was the best concentration of IPTG for expression of E3 scfv. Later, we used binding buffer with imidazole in different concentration as pre-eluting buffer in purification of E3 scfv and analyzed the bioactivity of purified E3 scfv by Western blotting assay. The result revealed that 10 mM was the best concentration of imidazole for pre-eluting buffer. Thus, we established a stable expression and purification procedure to get purified E3 scfv for futher study.The purified E3 scfv was coated immobilized as a target for screening a phage peptide library to get the specific phages which were able to bind with it. After four rounds of biopanning, 94 clones were picked up randomly and analyzed by phage ELISA assay, among which 24 positive clones were found. Amino acid sequences deduced from these 24 DNA sequences showed only one sequence, which was FTPY PLTAPVGS. We compared the amino acid sequence with that of Aβ_1-40 and found its VGS matched with Aβ_24-26 fragment. So, Aβ_24-26 was regarded as the epitope of E3 scfv.These experiments established good foundation for further study of E3 scfv on AD models and clinical trials.