Objective: To construct and express a single chain fragment of variety region (scFv) against β-site of amyloid precursor protein (APP), and evaluate the effect of scFv on α- and β-processing of APP.
Methods: The β-site specific scFv 2H10 was amplified and fused with signal sequence Igκ and myc tag by PCR to create the expression cassette, which was then subcloned into expression vector pcDNA3.1hyg⁺. The plasmid was transferred into CHO cells over-expressing Swedish mutation type human APP695 (APP695sw/CHO). The positive clones were identified by hygromycin B. The concentrations of Aβ40 and soluble APPα (sAPPα) in the conditioned media supernatant were measured by sandwich ELISA.
Results: The recombinant eukaryotic expression plasmids were identified by PCR, restriction endonuclease digestion and DNA sequencing. The expression of scFv was confirmed by Western blotting. When scFv 2H10 was stably expressed in APP695sw/CHO, the extracellular level of Aβ40 decreased by 30.4%, while sAPPα increased by 23.1%.
Conclusion: The APP β-site scFv 2H10 could inhibit the secretion of Aβ. Meanwhile, it might be favorable for the α-processing of APP. Hence it can be explored as an inhibitor of β-site APP-cleaving enzyme (BACE).