Preparation And Imunopharmacologcial Study Of A Self-assembling Aβ Epitope Vaccine Based On Clinically Therapeutic Enzyme

Vaccines are an important strategy for the prevention and treatment of major human diseases,and are also a major component of modern biotechnology drugs.Epitope vaccines with high antigen purity,strong targeting,few side reactions,and simple preparation have become the mainstream of modern vaccine development.However,the biological properties of small molecular weight,simple structure and single response mechanism have greatly limited its immunogenicity and affected the clinical development and application of this vaccine.Alzheimer disease(AD)is a lethal neurodegenerative disease with complex pathological mechanisms that cannot be effectively prevented.Recently,the immunization strategy targeting amyloid β(Aβ)has brought hope to the effective prevention and treatment of AD.Epitope vaccines and antibody drugs based on Aβ functional B cell epitope(Aβ1-15)are distributed in Pre-clinical and clinical research stages.Studies have revealed that how to effectively improve the immunogenicity and protective function of epitope peptides on the basis of ensuring immune safety remains a key issue in the development of A(3 epitope vaccines,and is also a common demand for epitope vaccine development.Therefore,based on the preliminary work of the research group,this paper is based on the fusion protein of L-asparaginase B(AnsB)and the universal HLA-DR reactive epitope(PADRE)as a vector designed and developed a novel Aβ epitope vaccine and expression preparation and preliminary immunopharmacological studies were performed on it.Firstly,the paper resuscitate culture and detectione the recombinant expression vector engineering bacteria of the novel epitope vaccine antigen protein AnsB-PADRE-Aβ1-15(APA)which designed and constructed by the research group.Furthermore,IPTG induce antigenic protein highly efficient soluble expression,and extraction,separation and purification of antigenic proteins by protein engineering techniques such as bio-enzymatic digestion,ultrasonication.salting-out precipitation.affinity column chromatography and gel filtration;again,study the relative molecular weight,protein purity,content,homologous self-polymerization form,antigenicity of antigenic proteins by SDS-PAGE,BCA,gel column chromatography,enzyme-linked immunosorbent assay (ELISA)and Western-Blot;on the basis of this,wild-type C57BL/6 mice were used as experimental subjects,and the evaluation was performed by anti-Aβ-specific antibody levels,antibody affinity and IgG subtypes in serum.Further comparative study of antigenic proteins with emulsified adjuvants(such as Freund’s adjuvant),granular adjuvants(such as aluminum hydroxide adjuvant)and nucleus Adjuvants(adjuvants such as CpG oligonucleotides),campare different forms of adjuvant efficacy of combination and selection of the most suitable vaccine from combinations.Finally,B6/J-Tg(APPswe,PSENdE9)/Nju(referred to as APP/PSN)transgenic mice were used as the pathological model of AD.The immunological pharmacological effects of the new vaccine were studied by animal immunity,humoral immunoassay and learning and memory evaluation based on Morris water maze.The results showed that the high-efficiency expression of antigenic proteins in the prokaryotic system was achieved by the cultivation and induction of engineering bacteria,and it was mainly expressed in soluble form in bacterial cytoplasm,which facilitated the correct folding and purification of antigenic proteins.The optimized protein purification process obtained the target antigen protein with purity greater than 95%,which confirmed that the relative molecular weight of the antigenic protein single subunit was about 42 kDa,the apparent molecular weight was about 164 kDa,and the antigenicity and accessibility of fusion epitope Aβ1-15 was maintained,revealing that the antigenic protein forms a homotetrameric molecule capable of tetravalently presenting the Aβ 1-15 epitope through the self-polymerization process of AnsB;further based on in vivo studies of wild-type C57BL/6 mice,The obtained antigenic protein exhibits different immune response intensity and mechanism when combined with different types of adjuvants,and the combination with Freund’s adjuvant has significant advantages in immunogenicity and humoral immune bias,and can induce specific humoral immunity neutralization Aβ neurotoxicity and targeted recognition of Aβ amyloid plaques;on this basis,the paper will an optimized combination of adjuvants was used to inoculate APP/PSN in AD transgenic model mice,and it was found that the novel antigen showed relative immune tolerance in a pathological model capable of endogenous expression of Aβ,while the neuroprotective Chinese medicine extract TCME-1701 can overcome tolerance,induce more intense Th2 anti-Aβ humoral immunity in pathological model,reduce amyloid deposition in brain of pathological mice,and improve the cognitive,learning and memory ability of model mice to some extent.In conclusion,based on the preliminary work of the research group,this paper successfully prepared a novel tetravalent Aβ epitope vaccine antigen protein based on the self-polymerizing pharmaceutical enzyme AnsB and the universal helper T cell epitope PADRE fusion protein vector,which proved that the antigenic protein can induce a safer and more effective Th2 anti-A(3-specific immune response in wild-type mouse and AD transgenic models,demonstrating the potential for effective prevention and treatment of AD,and reveals the feasibility and practicability of the self-polymerizing pharmaceutical enzyme AnsB and universal type helper T cell epitope PADRE fusion protein vector as a Th2 epitope vaccine vector,provides a new carrier supplement and technical solution for improving the immunogenicity of epitope vaccine,provides a candidate antigen to effective safe prevention and treatment for AD.and a potential helper enhancement strategy is provided for Aβ vaccines designed and developed based on the same immune mechanism.