Selection, Identification And Preliminary Study Of Affinity Maturation Of ScFv Specific Against Aβ_1-42 Oligomers

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease in the elder. The disease is characterized by memory impairment and cognitive obstacle. The current treatments of AD provide only symptomatic relief and are practically inefficient. Now, anti-Aβantibodies have been suggested as a potential therapeutic strategy for the treatment of AD.Genetic engineering made it possible to generate antibody without constant region which is termed scFv(single chain fragment variable) or Fab (Antigen binding fragment). ScFv is a smaller functional unit remaining antigenic specificity and affinity. Compared with other antibodies, scFv has many advantages, smaller size, reduced immunogenicity, stronger penetrating power, rapid localization and being easily cleared up from the normal tissue. Meanwhile, scFv can be produced in large scale in bacteria or yeast and manipulated by genetic engineering. Therefore, developing scFv is of great significance in the areas of AD diagnosis and therapy.To construct the human single-chain Fv (scFv) antibody library by phage display technology and obtain the specific scFvs (single-chain fragment variable) against soluble Aβ1-42 (Amyloid-beta ) oligomers by screening the human scFv library, peripheral blood lymphocytes (PBL)were separated from ten healthy donors, and the total RNA was extracted from the PBL. The variable heavy(VH) and variable light(VL) genes were amplified by RT-PCR and then the scFv was obtained through SOE-PCR. The scFv fragments were cloned into the vector pCANTAB5E and electroporated into competent E.coli TG1 cells, and then a scFv phage display library containing 2.5×109clones was gained. The recombinant phagemids were rescued by reinfection of helper phage M13K07. Recombinant phages specific for Aβ1-42 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. The soluble scFv antibodies were expressed successfully, and displayed specific binding to Aβ1-42 trimer and protofiber by SDS-PAGE and Western blot analysis.The binding affinity of soluble scFv antibodies to Aβ1-42 oligomers was 9×10-6 mol/L. For the purpose of affinity maturation, the VH and VL gene fragments were cloned into the pComb3C/cFab vector, and transformed to competent E.coli TOP10. SDS-PAGE and Western blot analysis confirmed that the soluble B19-Fab fragments were expressed successfully, and preserving the binding affinity to Aβ1-42 oligomers. The preparation of specific scFv against Aβ1-42 oligomers can be used further in the therapeutic research on AD.