Study On Preparation Technology Of Protein Vaccine For Alzheimer’s Disease

Dementia has been known as one of the world’s current"epidemics"and is considered to be the fourth killer for the health of the elder people.The most common cause of Dementia is Alzheimer’s disease.Alzheimer’s disease（AD）is a progressive age-related neurodegenerative disorder which occurring in the elderly and early age of primary encephalopathy,mainly lead to the dysfunction in the performance of central nervous system..Currently,the deposition of amyloidβ（Aβ）in the brain is regarded as the central pathological hallmark of AD.Thus,immunotherapy targeting Aβclearance may be the most promising therapeutic options for AD.Norovirus particles P having three cyclic structure,very suitable for insertion of foreign epitopes;Furthermore,norovirus particles P can be formed in vitro autonomous means 24 mer form,is a very good antigen displaying internet.In this study,we used Aβ1–6,which only contains B-cell epitopes as a novel immunogen,and P particles as the vaccine platform.The three copies of Aβ1–6 gene were inserted into the loop domain of the P particles.And then we obtained the plasmid pET26b-PP-3copy-Aβ1-6-loop123.The recombinant P particles were expressed in E.coli BL21 and the protein expression conditions were optimized.The optimum conditions of protein expression were as follows:for lab test,the inducing temperature was 16℃,the concentration of IPTG was 0.5mmol/L,and the inducing time was 12 h;moreover,for the excellent conditions of fermentation tank,the optimum inducing time was 11 h.After the optimization,the yield of the protein expressed in soluble expression system was increased from 1.5 mg/L to 8 mg/L in the test.The protein content of the protein expressed in inclusion body expression system was increased from 60 mg/L to 300 mg/L.What’s more,the polymer form and particle size of the target protein were identified by Superdex^TM200,transmission electron microscopy and particle size analyzer.The result showed that,the protein had three polymer forms:24-mer,12-mer and 2-mer.The proteins from the soluble expression system were mainly in the form of 24-mer PPs,and the size of protein was about 24 nm.In contrast,proteins expressed in inclusion bodies were mainly in the form of 12-mer PPs,and the size of protein was about 16 nm.Finally,the immunogenicity and safety of the protein vaccine were detected by immunization in mice.The results showed that PP-3copy-Aβ1-6-loop123 could efficiently stimulate Aβantibody titers without inducing a T-cell-mediated immune response,and no meningoencephalitis was observed,suggesting PP-3copy-Aβ1-6-loop123 was a safe AD vaccine.This study laid foundation for the industrial production of this AD vaccine PP-3copy-Aβ1-6-loop123.