Isolation and identification of a human single chain Fv antibody against amyloid-beta 1-42 soluble oligomers from a human phage display library.
- Author:
Fuxiang BAO
1
;
Jinsheng HE
;
Guifang CAO
;
Fan YIN
;
Xin WANG
;
Siyuan PANG
;
Ying ZHANG
;
Tao HONG
Author Information
1. College of Life Sciences & Bioengineering, Beijing Jiaotong University, Beijing 100044, China.
- Publication Type:Journal Article
- MeSH:
Amyloid beta-Peptides;
genetics;
immunology;
Antibody Specificity;
immunology;
Humans;
Peptide Fragments;
genetics;
immunology;
Peptide Library;
Single-Chain Antibodies;
genetics;
immunology;
isolation & purification
- From:
Chinese Journal of Biotechnology
2009;25(8):1195-1203
- CountryChina
- Language:Chinese
-
Abstract:
To get specific scFv (Single-chain fragment variable) antibody against soluble Abeta1-42(Amyloid-beta) oligomers, we constructed a human single-chain Fv (scFv) antibody library by phage display technology. Using RT-PCR, we amplified the variable heavy (VH) and variable light (VL) genes from peripheral blood lymphocytes (PBL). Then we obtained the scFv fragments through SOE-PCR, and the scFv fragments were cloned into the vector pCANTAB5E and electroporated into competent Escherichia coli TG1 cells. Consequently, a scFv phage display library containing 2.5 x 10(9) clones was constructed. The recombinant phagemids were rescued by reinfection of helper phage M13K07. Recombinant phages specific for Abeta1-42 oligomers were enriched after four rounds of biopanning and the antigen-positive clones were selected from the enriched clones by phage ELISA. Positive clone B19 was used to infect E. coli HB2151 to express soluble scFv antibody. SDS-PAGE and Western blotting analysis showed that the soluble scFv B19 antibody was expressed successfully and could bind specifically to Abeta1-42 trimer and protofiber. The specific scFv against Abeta1-42 oligomers can be used in the therapeutic research on Alzheimer's disease.
