[Cloning and expression of human single-chain Fv antibody against amyloid beta peptide involved in Alzheimer's disease].Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2003 Oct; 25(5):557-62.ZY
To screen out the specific antibody clones against amyloid beta peptide 40, and clone the antibody gene and express it in a bacterial system, so as to provide a solid basis for novel diagnostic and therapeutic methods for Alzheimer's Disease.
beta amyloid peptide 40 was bound on the solid surface of Nunc plates as antigen to screen the binding clones from a phage-display human single-chain Fv antibody library. After five rounds of bio-panning, the host E. coli TG1 was infected with eluted filamentous phage from the last turn of selection. 55 well-separated colonies were picked randomly from the plates and the specific positive clones were identified by ELISA test. The single-chain Fv antibody gene was sequenced and their amino acids sequence was deduced. The scFv antibody gene was sub-cloned into a protokayotic expression vector pGEX-6P-1 and transformed into bacteria strain BL21 to express the glutathione-S-transferase (GST) fusion single-chain antibody.
ELISA test showed that 33 of the 55 clones could bind amyloid beta peptide 40 and 10 of the 33 clones could be inhibited by amyloid beta peptide 40 itself to below 50% of its original binding activities. Five of the 10 clones could also be inhibited by amyloid beta peptide 1-16 to the same level, which meant that the binding epitope of the antibody from the 5 clones was between first to sixteenth amino acids at amino-end of amyloid beta peptide 40. DNA sequencing data demonstrated that the gene of the single-chain antibody specifically against amyloid beta peptide 40 was consisted of 768 bp and the deduced amino acids sequence confirmed its typical antibody structure. The complement determinant regions and framework regions were discriminated empirically. After cloning the antibody gene into a protokayotic system, the GST fusion antibody was expressed as the expected size.
After five rounds of bio-panning and subsequently serial ELISA testing, the specific antibody clones against amyloid beta peptide 40 were screened out successfully. The antibody gene DNA sequence and amino acids sequence were analyzed and confirmed. The fusion antibody was expressed as expected in the bacterial system.