The Study On High Expression, Purification And Oligomer Formation Of Alzheimer's Disease Aβ_1-42 Polypeptide

Alzheimer's disease, AD is nerve degenerative disease with progress memory loss and aphronesia. It's pathological characteristics areβstarch-like fibers(AβPeptide ,Aβ) were precipitated in the brain nerve cells, and then develop senile plaques ( SP )and Neurofibrillary tangles, NFT were appeared Cellula nervosa. The specific patho-mechanism is still unknown as yet. However, many research evidences showed Aβoligomer plays a very important role in the development of AD. Accordingly , the study of Aβoligomer might have potential therapeutic efficacy to Alzheimer's disease.Purpose:Economy, High performance and Easy preparation are the prerequisite and foundation in the immunotherapy of Aβoligomer. Our research group got the Aβ_1-42 polypeptide in the way of prokaryocyte expression. After that,the sequestration laminar analysis and purification were carried out to find the condition and approach of Aβoligomer formation. So that it can provide economic materials (Aβoligomer) to produce monoclonal antibody forward to immunotherapy. Meanwhile, prokaryotic expression and recombination protein purification were carried out in two plasmids which both contain Aβ42 to find the more suitable expression plasmid.Approach:Construct recombination plasmid of PET30a and Aβ_1-42 ,and transfer the recombination plasmid to the Escherichia coli BL21 to express,and then, purify the protein by Ni-NTA Superflow sequestration on the condition of urine denaturation. After that, dissolve the purified Aβ_1-42 polypeptide in F12 medium with HFIP and DMSO. Setting up several conditions to fumble the law of oligomer formation in solution of Aβ_1-42-F12. Compare with other approaches, we primarily found the approach and condition to precipitate Aβ_1-42 oligomer.Result:After many repetatur experiments in different conditions,high express and purification from recombination were selected. In addition, molecular weight and Immunologic competence are exactly the same. Finally, we brought up the polypeptide,in the temperature of 37℃,as monomers decreased in the third and fourth day, the oligomer were formed,and the oligomer grew as the days went by.