[Isolation and identification of a human single chain Fv antibody against amyloid-beta 1-42 soluble oligomers from a human phage display library].
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.
Bao
Fuxiang
F
College of Life Sciences & Bioengineering, Beijing Jiaotong University, Beijing 100044, China.
He
Jinsheng
J
Cao
Guifang
G
Yin
Fan
F
Wang
Xin
X
Pang
Siyuan
S
Zhang
Ying
Y
Hong
Tao
T
D016229:Amyloid beta-Peptides / Q000235:genetics* / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D006801:Humans; D010446:Peptide Fragments / Q000235:genetics* / Q000276:immunology*; D019151:Peptide Library; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*
D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; D019151:Peptide Library; D057127:Single-Chain Antibodies; C075222:amyloid beta-protein (1-42)