Expression And Purification Of β-Amyloid Peptides (Aβ) And The Fuction Of Aβ-induced Antibodies

Alzheimer's disease (AD) is a progressive neurodegeneration and the most -common form of dementia in the elderly. The disease is characterized by massive cell loss, especially of cholinergic neurons, deposition of fibrillar amyloid-β(Aβ) peptides as senile plaques, and intracellular accumulation of hyper-phosphorylated tau protein as neurofibrillary tangles. Although AD pathogenesis is complex and remains unclear, aggregation of Aβpeptides is considered to be the first known event in a complex cascade eventually leading to neurodegeneration.Aβis a hydrophobic peptide contains 42 amino acid residues. There are three major epitopes on the Aβbeta peptide: (i) antibodies to the N-terminal epitope of the Aβpeptide bind to aggregated amyloid beta peptide in vitro as well as cerebral and vascular deposits in vivo and APP ; (ii) antibodies specific for the central region of the Aβpeptide bind to APP but not to aggregated amyloid beta peptide in vitro, amyloid plaques or vascular amyloid deposits ; (iii) antibodies specific for the C-terminal region have been less well studied but reported to lack a therapeutic effect in APP-transgenic mice. Some reports indicated that epitpoes on C-terminal region induce T-cell mediated encephalitis in active immunotherphy. Hence, it seems that different epitode can induce different intensity of humoral immunity. Furthermore, the ability of these antibodies differs in interrupting Aβaccumulation and alleviating the cell-toxicity of Aβpeptide. pan HLA DR binding Epitope (PADRE) is a synthetical peptide which can reinforce they response between antigen presenting cells and TCR. Tetanus toxin epitopes (TT), developed form cl.tetani, is also a widely used Th-Cell epitope. Therefore, both of PADRE and TT can enhance the immunoreactions of bodies. ISS in this thesis contained PADRE and TT .To take these two factors into account, we constructed 6 kinds of recombining vector which can express the following peptides: GST-Aβ28,GST-Aβ35,GST-Aβ42,GST-IAβ28,GST-IAβ35,GST-IAβ42. Six kinds of peptides, GST-Aβ28,GST-Aβ35,GST-Aβ42,GST-IAβ28,GST-IAβ35,GST-IAβ42 were used as antigens to inject mouse. ELISA detected the titers of all antibodies towards Aβ42. The results revealed that the anti-GST-Aβ42 serum had the highest titer, the anti-GST- Aβ28 serum ranked the second one and the anti-GST- Aβ35 serum was the lowest one. Although the addition of ISS sequence to the N terminus of two antigens (GST- Aβ28,GST- Aβ35) could reinforce their immunoresponse, anti-GST-IAβ42 serum decreased abnormally. Moreover, Anti-GST-Aβ42 showed the tiptop ability in interrupting Aβaccumulation and alleviating the cell-toxicity of Aβ, anti-GST- IAβ28 serum is the second one,anti-GST- IAβ35 serum was the lowest one. Considering the cell toxicity of Aβ,cerebral hemorrhage presumably from vessels with Aβdeposits caused by the N-terminal-specific anti-Aβand T-cell mediated encephalitis induced by C-terminal region of Aβ, appropriate antigens or antiboies should be chosen cautiously in special conditions.