Preparation Of Anti-Aβ Single Domain Antibody/Nanoluc Fusion Protein And Preliminary Verification Of Its Characteristics

Among neurodegenerative diseases,Alzheimer’s disease(AD)is the most common.It does not show obvious characteristics in the early stage of the disease.Once the patient has behavioral abnormalities such as severe memory loss,his brain structure will appear irreversible.Amyloid beta(Aβ)is one of the main causes of brain changes and is considered to be closely related to the pathogenesis of Alzheimer’s disease.In recent years,researchers usually use Aβ biomarkers as An important target for the diagnosis and treatment of AD.In this experiment,the target gene Nanoluc was first amplified from the nanoluciferase pNL1.2 vector by PCR technology,and then connected to the vector pET-25b(+)containing the specific single-domain antibody VHH gene to construct the recombinant plasmid;The constructed recombinant plasmid was transferred to E.coli BL21(DE3)competent cells to induce expression of the recombinant protein and optimize the expression conditions of the recombinant protein.The expressed product was analyzed by SDS-PAGE,and the recombinant protein was purified by nickel column,and the recombinant protein concentration was determined using the kit for protein concentration determination by BCA method.The negative control plasmid pET-25b(+)-Nanoluc without VHH gene was constructed using the same procedure.The binding activity of recombinant protein pET-25b(+)-VHH-Nanoluc to Aβ oligomers was identified by indirect ELISA and bioluminescence one-stepELISA.The expressed recombinant protein was injected into the mouse brain through a brain stereotaxic instrument,the substrate was injected into the tail vein,and the binding of the recombinant protein to Aβ in the model mouse brain was detected by a live imager.The following are the results of the experimental study:(1)The recombinant plasmid pET-25b(+)-VHH-Nanoluc and the negative control plasmid pET-25b(+)-Nanoluc were successfully constructed.The recombinant proteins expressed in E.coli BL21(DE3)competent cells were 37 kDa and 22 kDa in size respectively,as expected.The recombinant protein pET-25b(+)-VHH-Nanoluc expressed the highest protein concentration of 216.85 μg/m L when lactose was used as the inducer and the induction temperature was 16 ℃ for 12 h.(2)The recombinant protein has high binding activity to Aβ oligomers detected by indirect ELISA and bioluminescence one-stepELISA.By injecting the protein into the brain of mice using a brain stereotaxic instrument,and injecting the substrate into the tail vein,a higher fluorescence value was detected by the in vivo imager.The binding ability of Aβ in the brain of the model mice,the negative control groupwas injected with recombinant protein pET-25b(+)-Nanoluc,no fluorescence value was detected,which confirmed that it was not the non-specific binding between the enzyme and the substrate.In this study,a class of proteins that can specifically bind to Aβ oligomers and can be imaged with Aβ in the brain of AD model animals has been prepared,which provides a new idea for the follow-upstudy of Aβ-targeted therapeutic regimens and detection of therapeutic effects.