Construction, Expression And Biological Function Of Single Chain Fragment Variant Against Aβ Oligomers

Objects:Alzheimer’s disease (AD) is a kind of common neurodegenerative disease which causes dementia in aged people and there are no effective treatment methods. Aβ oligomers are the causative agent in the early stage of AD. On the basis of Aβ oligomers-specific monoclonal antibody A8established in our group, we are going to construct and experess the A8derived single chain fragment variant (scFv), and verify the biological function.Methods:In order to construct the scFv gene, the variable regions of light and heavy chain from A8were used as the template, and splicing overlap extension polymerase chain reaction (SOE-PCR) was exploided to get the scFv. As for the linker sequence, we used (G4S)3or p2A sequenze. Prokaryotic expression system was constructed just to verify the biological activity of scFv by a quick method. Then we constrcted the eukaryotic expression vectors. Hela and CHO cells were transfected with Lipofectamine2000and screened with hygromycin B. The supernanent was detected by indirect ELISA and dot blot, and its function was identified by cell ability assay and ultra pathology analysis.Results:We got three scFv gene fragments, VL-(G4S)3-VH、VH-(G4S)3-VL and VL-p2A-VH. The prokaryotic expression vectors containing the first two gene fragments were constructed. The target scFv of30kD protein were expressed and purified through nickel affinity chromatography. Western blot results showed that its molecular weight is correct; The indirect ELISA results identified the titer of VH-(G4S)3-VL and VL-(G4S)3-VH is1:32000and1:8000respectively. We have construced three eukaryotic expression vectors (pSecTag2/HygroA-VL-(G4S)3-VF、 pSecTag2/HygroA-VH-(G4S)3-VL and pSecTag2/HygroA-VL-p2A-VH). We obtain two stains of stable lines secreting scFv, Hela-VL-p2A-VH and CHO-VL-(G4S)3-VH. Indirect ELISA and dot blot showed that the "supernatant specifisically recognized Aβ oligomers; experiments in vitro showed that the supernatant of such cell lines could block and inhibit the cytotoxicicity induced by Aβ oligomers. Furthermore, the expression levels of cell line Hela-VL-p2A-VH is relatively higher than CHO-VL-(G4S)3-VH.Conclussion:We have successfully constructed Aβ oligomers specific scFv expression vector, which was correctly expressed by prokaryotic and mammalian systems; Two stable cell lines secreting scFv were established; The supernatant could specifically recognize Aβ oligomers, and inhibit the cytotoxicity generated by A(3oligomers, which paved the way to further application of AD immunotherapy.