Study On Recombinant Genetic Engineering Subunit Protein Vaccine Of Alzheimer's Disease

As a neurodegenerative disease,Alzheimer's disease mainly leads to the decline of cognitive ability and memory ability,bringing severe physical and mental stress to the patients and financial burden to the families and society.At present,with an increasing morbidity,Alzheimer's disease have caused critical concern around the world and variety of drugs are being developed for the prevention and treatment of this disease.There are many views on the pathogenesis of Alzheimer's disease.The highest degree of recognition is the amyloid cascade hypothesis,which suggests that the abnormal degradation of APP protein leads to the production of a large amount of soluble A?42 or A?40 peptide in the brain of the patients,and its further aggregation leads to the accumulation of a large number of amyloid plaques in the brain,causing tau to form excessive entanglement of nerve fibers,resulting in imbalance of ions and then cause neuronal cell necrosis.A large number of studies have shown that the accumulation of excess A? in the body is an important possible cause of AD pathogenesis.The accumulation of A? can cause damage to neurons and lead to neuronal declination,resulting in memory and cognitive dysfunction.Thus,active and passive immunization targeting A? toprevent and treat AD emerges.The AN-1792 Vaccine clinical trial failure was due to toxic T cell immune response trigged by T cell epitopes of A?42 peptide.In this study,the recombinant fusion antigen 12 A?15-TF was constructed by repeated 12 copies of human A?42 B cell epitope with the auxiliary of T cell epitope.This recombinant fusion antigen not only can avoid the side effects of the immune response and ensure the safety of the vaccine,but also may specifically remove the toxic A? oligomers to achieve the purpose of prevention and treatment of AD.The gene encoding 12A?15-TF antigen was cloned into the prokaryotic expression vector pTIG-Trx,and then the recombinant protein expressed in prokaryotic was purified.The immunogenicity of the vaccine was determined after immunized of C57BL/6 micewith Al adjuvant.The 6A?15-TF recombinant protein previously prepared was used as a parallel control.The results showed that after four immunizations,the mice in the immunized group produced very high specific anti-A?42 antibody,and the highest antibody titer was about 1: 40000.The results of antibody subtypes showed that the antibody subtype produced by these two vaccines were mainly IgG1 accompanied by a certain amount of IgG2 b,and the immune response was mainly Th2 humoral immune response.The results of lymphocyte proliferation showed that the C57 BL / 6 mice were immunized with the T lymphocyte immune response against A?42.These results suggest that the vaccine may be able to specifically neutralize or elimination the A?42 molecule in the body for the treatment or alleviationof Alzheimer's disease.Therecombinant protein 6A?15-TF and 12A?15-TF were used to immune 3× Tg-AD mice separately combined with Al adjuvant.After five immunizations,a high level of antibody against A?42 peptide was also produced in 3 × Tg-AD model mice and maintained in vivo for a longer period of time.The results of the antibody subtype assay showed that the vaccine immunizing 3 × Tg-AD mice produced a high IgG1 immune response accompanied by a certain amount of IgG2 b reaction,similar to the previous results with C57BL/6 mice.Both vaccines induced Th2 type immune response without causing Th1 immune inflammatory response.The above experiments showed that the recombinant vaccine 12A?15-TFproduced a strong Th2 type humoral immune response similar with 6A?15-TF,suggesting that the vaccine may not produce A?42 specific Th1 immune responsein clinical trials,thus,have higher clinical safety.The behavioral evaluation experiments were performed in 3 × Tg-AD mice at 20 months of age after the vaccination.The results showed that the learning and memory performance of mice have been improved at a certain level.The brain tissue protein was extracted for the detection of AD pathology-related proteins.The results showed that some synaptic proteins in the mice after treatment had some positive changes compared with the untreated mice.The expression of Dynamin1 protein in the mice with AD decreased.And the expression of Dynamin1 protein in the immunized group was significantly higher than that in the control group.The expression of PSD-95 protein was negatively correlated with the pathology of AD model mice.Compared with the control group,the level of PSD-95 protein was significantly up-regulated,indicating that the recombinant epitope chimeric vaccine was helpful towards protecting the mouse brain neurons.In addition,the contents of soluble and insoluble A?40,A?42,and Tau in brain tissue of 3 × Tg-AD mice were measured.The results showed that the content of soluble A? and soluble tau in mice immunized with 12A?15-T was significantly decreased compared with mice immunized with 6A?15-T,however no significant difference was found when it comes to insoluble Tau.In conclusion,the 12A?15-TF recombinant protein vaccine has a good immunotherapy effect on AD,which can reduce the pathological changes of AD and improve the behavioral ability of the mice,suggesting that this vaccine can be used as a candidate vaccine for immunotherapy or prevention of Alzheimer's disease.