Expression Of Anti-Aβ Single Chain Variable Fragment In Baculovirus And Effect On Aβ Aggregation And Degradation In Vitro

Alzheimer’s disease (AD) is the most common age-related dementia and affects35.6million people worldwide. The misfolding and aggregation of P-Amyloid peptide (Aβ) is one of the most important pathological characteristics of AD. An Aβ oligomer specific monoclonal antibody (MAb), designated A8, has been established in our group and has been proved to have potential therapeutic effect on AD. In order to study the mechanism by which how this antibody works, we plan to construct and express single chain variable fragment (scFv) which is derived from A8, and then try to investigate how Aβ aggregation and degradation would be regulated by scFv.Methods:i) Expression, purification and identification of scFv. The primers were designed according to the sequence of variable region of light chain (VL) and heavy chain (VH) of MAb A8, and the His tags were introduced to amino and carboxyl terminus respectively by PCR. Then the rBacmids were constructed and transfected into Sf9cells to express the scFvs. The expressed scFvs were purified by Ni-NTA Agarose. The expression and purification of scFvs were identified by Western-blot, and the ability of scFvs to recognize Aβ was identified by dot blot and indirect ELISA. ii) Establishment and optimization of Aβ aggregation model in vitro. Optimized experiment condition was chosed to grow Aβ fibril and overcome the de novo peptide aggregation. Then samples were collected from reaction mixture at different time points, and all the samples were detected by transmission electron microscopy (TEM) after negative staining, iii) Bidirectional interference effect between scfv and Aβ. Firstly, the anti-AP scFv and Aβ monomer were incubated in optimized reaction system, with the scFv buffer and A8as control groups. The length of Ap fibril corresponding to some certain time points were detected by TEM. And then the dose-dependent assays were performed to prove the specific role of scFv. Finally, the A8scFv were incubated with mature Aβ fibrils (48h sample) to study the degradation effect of scFv.Results:i) Anti-AP scFvs with His tags were solubly expressed successfully in Baculovirus expression system, and the results of western blot showed that the molecular weight of scFvs were about30kDa as we expected. The results of dot blot and indirect ELISA showed that scfv could specifically recognize Aβ oligomers. ii) Aβ aggregation model in vitro was established and the borate buffer (pH8.5) was showed to be the best reaction buffer to grow Aβ fibril. Aβ was easy to aggregate as soon as it were incubated in37℃and the length of fibrils was about500nm after incubation of24h, then the fibrils were elongated to more than1000nm after48h. The length and the diameter of fibrils were increased significantly with time, and after96h the tangle-like Aβ aggregation formed in buffer. iii) Inhibition of Aβ on-pathway aggregation by the A8scFv. In the experiment group with scFv (p<0.01), the length of Aβ fibril was obviously shorter compared with buffer and control groups, which showed that scFv can inhibit the formation of Aβ fibril. We then incubated pre-formed Aβ fibril with anti-Aβ scFv and found that the length of fibrils were reduced significantly (p<0.01) compared with the experiment group without scFv. This indicated that anti-Aβ scFv has the ability to degrade mature Aβ fibrils. In dose-dependent assays, anti-Aβ scFv could inhibit elongation of Aβ fibril when the Aβ/svFv molar ratio was1to10(p<0.01), but there was no significantly effect in1-fold and5-fold experiment groups.In conclusion, anti-Aβ scFv which is expressed by Baculovirus expression system can inhibit formation of Aβ fibrils and degrade the mature Aβ fibrils in dose-dependent manner. This study lays the foundation for further study of the mechanism of AD therapeutic strategy.