The Preparation And Evaluation Of Anti-Aβ12-35 Nanobodies

Alzheimer’s disease (AD) is a common form of dementia. The pathogenesis of AD is still not conclusive, numerous studies have shown that AP are important factor initiated AD pathology. Therefore, the development of inhibitors or drugs, which can suppress Aβ aggregation or induce degradation of Aβ fibrils, has become a hot research area in AD immunotherapy. At present, antibodies for AD immunotherapy are humanized monoclonal or polyclonal antibodies and small single chain antibodies etc. Compared with the antibodies above,the advantages of nanobodies like low preparation cost and easy to cross the blood-brain barrier make them get more and more attention. In addition, the existing research shows that the N terminal of Aβ doesn’t involve in aggregation, and the antibodies recognized N terminal of AP can cause strong inflammatory reaction so that the clinical trial has to be stopped; while the middle fragment of Aβ is the key site and toxicity part in Aβ aggregation, it can easily form a β-sheet structure, which is main structure features of Aβ aggregation. Antibodies which can recognize Aβ aggregation section its inflammatory reaction, is lower relatively, so screening of nanobody that specifically binding Aβ12-35 will be important in inhibiting aggregation and exploring the mechanism of aggregation, In this research, the middle fragments of full-length Aβ, Aβ 12-35, was chosen as antigen for nanobody selection, and nanobodies were prepared with E.coli expression system, then we discussed their impact on Aβ aggregation. The research contains what listed below:(1)Nanobody sequences selection. Aβ12-35 monomer was used as antigen and adopted site-directed immobilization, after experienced four-round selection and detected by ELISA,4 fragments, DP4, DP6, DP18, DP36 were isolated respectively from a Aβ42-immune library.(2)The expression of nanobodies. The recombinant vectors was constructed by PCR and double enzyme digestion etc. After gene sequencing, the positive recombinant plasmids of nanobody were transformed into host bacterium E.coli Shuffle T7 and E.coli Origami 2(DE3) and all of them were soluble expression.The purified nanobodies(S-DP4, O-DP4, S-DP6, O-DP6)showed high binding activity which detected by ELISA. Biacore results showed that the affinity constant of S-DP6 was 10-7, the others were 10-6, which were basically consistent with ELISA results.(3)Function of nanobody. ELISA results showed that nanobody S-DP6 can not only recognize Aβ monomer and aggregation, but also the monomers and aggregation of Aβ40 and AP42. In vitro the ThT fluorescence and electron microscopy confirmed that S-DP6 can inhibit Aβ12-35 monomers aggregation and fibrils formation effectively.In conclusion, nanobodies in this research can recognize a variety of AP and effect Aβ aggregation, supplying useful research materials for study of mechanism of AD caused by Aβ aggregation.