| Alzheimer’s disease(AD)is one of the most common neurodegenerative diseases.Genetic factors,environmental factors,and age are major factors in the occurrence and development of AD.About one in every 30 people aged 65 or older has dementia.In view of the increasing aging trend in the world today,it is bound to place higher demands on our society and economy.Therefore,it is increasingly urgent to actively develop drugs for the treatment of AD.The main pathological hallmark of AD is extracellular beta-amyloid deposition or amyloid plaque(Senile plaque,SP)and intracellular neurofibrillary tangles(Neurofibrillary tangles,NTFs).Amyloid plaques are mainly formed by the aggregation of Aβ42 produced by amyloid precursor protein(APP);neurofibrillary tangles are mainly formed by aggregation of hyperphosphorylated Tau protein(microtubule-associated protein).Currently,the main treatment for AD is immunotherapy targetingβ-amyloid based on theβ-amyloid hypothesis.It was found that 43,226,241 and 244 sites in ZZ6,a single-chain antibody against Aβ42 oligomers obtained in the previous study,are key sites based on the conformational simulation and molecular docking between ZZ6 and Aβ42 molecules,these sites are involved in Aβ42 binding,and formed a annular binding center,but there is no effective hydrogen bond between the two molecules because the steric hindrance is too large.In this thesis,amino acid mutations G43S,T226E,G241E,K244H for these four key points were designed,and the properties and functions of ZZ6 and its mutants were analyzed and studied.This thesis has obtained the following research results:(1)Three mutants of single-chain antibody ZZ6 were obtained by whole plasmid PCR or nested PCR,which were ZZ6-2-1(G43S),ZZ6-2-(1-2)(G43S,T226E),ZZ6-2-(1-3)(G43S,T226E,G241E,K244H).These single-chain antibodies were prepared by genetic engineering expression and purification.(2)The binding capability of four single-chain antibodies[ZZ6,ZZ6-2-1,ZZ6-2-(1-2),ZZ6-2-(1-3)]to Aβ42 oligomers was determined by indirect ELISA.The value of ZZ6 was 3.1nM,of ZZ6-2-1 was 2.1 nM,of ZZ6-2-(1-2)was 8.5 nM,and of ZZ6-2-(1-3)was 1.1 nM,indicating that they effectively binding Aβ42 oligomers at nanomolar level,in which the binding capability of ZZ6-2-(1-3)to Aβ42 oligomers is significantly higher than the other three.(3)Binding affinity constants(equilibrium dissociation constant,K_D)of four single-chain antibodies[ZZ6,ZZ6-2-1,ZZ6-2-(1-2),ZZ6-2-(1-3)]to Aβ42 oligomers were determined by competitive ELISA.K_D(ZZ6)was 3.1×10-6 M,K_D(ZZ6-2-1)was 3.0×10-6 M,K_D[ZZ6-2-(1-2)]was 3.42×10-6 M,and K_D[ZZ6-2-(1-3)]was 3.32×10-6 M,indicating that these antibodies exhibited similar binding affinity Aβ42 oligomers.(4)It was found by ThT-F,Western Blot and HPLC analysis that both ZZ6 and ZZ6-2-(1-3)can effectively inhibit the aggregation of Aβ42 and induce the depolymerization of Aβ42aggregates to some extent,but the effect of ZZ6-2-(1-3)is more pronounced.(5)It was found by MTT assay that both ZZ6 and ZZ6-2-(1-3)can effectively inhibit the cytotoxicity of Aβ42 oligomers,and the effect of ZZ6-2-(1-3)is more significant,and their protective efficacyis for target cells were also reflected in the morphology and motility of the target cells.(6)The results of HPLC analysis showed that ZZ6-2-(1-3)showed a certain effect of promoting the degradation of Aβ42.It was speculated that ZZ6 formed a catalytic centre-like conformation,showing a certain catalysis potential after mutation of G43S,T226E,G241E and K244H,which was also a manifestation of the evolution of ZZ6 function.The above research results of this thesis provide a theoretical basis and practical guidance for the development of more effective drugs to promote Aβ42 metabolism and anti-Aβ42oligomeric cytotoxicity. |
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