The Expression And Purification Of Human Aβ42 And Biopanning Of Nanobodies Anti-Aβ42

Metabolic abnormalities and accumulation of amyloid beta peptide 42 (Aβ42) are the earliest characteristics in the pathogenesis of Alzheimer’s disease (AD). So it’s significant to establish a method to detect the concentration and the aggregation of Aβ. To research the early diagnosis and targeted treatment of AD, nanobody, with high specificity and high affinity to Aβ, is a kind of promising and vigorously researched material. However, some problems are still in existence. Firstly, in most traditional methods, antigen is coated through hydrophobic interaction. This not only leads to poor binding ability but also increases the risk of protein morphology change. Secondly, almost all existing Aβ antibodies can only identify the primary structure of Aβ42, but can’t distinguish different conformations of various aggregates. In addition, synthetic Aβ is expensive and usually heterogeneous. Comprehensive consideration, in this thesis we have completed the followings:first we directly expressed and purified human Aβ42 and that with sulfydryl tag in the N terminal (C-Aβ42), providing good materials for the screening of nanobodies. Then the protein was used as target molecule to screen anti-hAβ42 nanobodies from immune phage display library in the method of site-directed immobilization. The main results are as follows:(1) The preparation of Aβ42 and C-Aβ42. First, recombinant plasmids of pET23a-Aβ42 and pET23a-C-Aβ42 were constructed and synthetized. Then, the two proteins were expressed as inclusion bodies in E.coli BL21 (DE3). The conditions of expression were optimized. After washing and being pretreated with HFIP, the inclusion bodies were purified by a one-step purification of DEAE anion exchange. Aβ42 and C-Aβ42 with high purity (higher than 95%) and relatively high yield (about 40 mg/L culture) were obtained. At last, the purified proteins were dialyzed, dried and kept at-80℃.(2) The characterization of Aβ42 and C-Aβ42. The proteins were pretreated with HFIP and resuspended in solutions as needed. In the assay of Tricine-SDS-PAGE and MALDI-TOF, AP42 and C-AP42 samples showed the expected size and high purity. The results of CD and ThT fluorescence indicated formation of a P-sheet-rich secondary structure. Moreover, the two proteins exhibited good immunogenicity and strong neurotoxicity on hippocampal neuron cells based on the analysis of dot-blot and MTT assay. The results above suggested that the method we developed would be useful in obtaining a large quantity and high bioactivity Ap42 and C-AP42 for the screening of nonobodies.(3) The screening of nanobodies from immunized phage display libraries in the method of site-directed immobilization. Proteins above were used as antigens and were covalently bonded onto maleimide-modified surface plate by sulfhydryl groups. Then the screening process was developed. Compared with the traditional coating method, site-directed immobilization was more conducive to enrich nanobodies with high activity. At the same time, the influence of pH and reducing agent was observed, indicating that pH 6.95 and the reducing agent would benefit the exposure of sulfhydryl groups and the immobilization of antigens. After the assay of Phage-ELISA and sequence alignment, we finally obtained 8 genes of anti-Aβ42 nanobodies from 28 highly active monoclonal phages.