| Alzheimer’s disease(AD), a neurodegenerative syndrome, is a major cause of elderly dementia. The complications of AD are the most common cause of death in the elderly. It is characterized by memory impairment, cognitive obstacle and mental disorder. Amyloid-beta(Aβ) protein that is accumulating extracellular in the AD brain and building cortical senile plaques and hyperphosphorylated Tau protein that is aggregating intracellular in the AD brain and building neurofibrillary tangles are the main neuropathological marks of AD. Several hypotheses have been put forward on the basis of the various causative factors in order to explain this disease, such as the cholinergic hypothesis, Aβ hypothesis, Tau hypothesis and inflammation hypothesis. Recently it has been shown that the Aβ hypothesis is commonly used. However, it does not account for the complex pathophysiology of this incapacitating disease. Aβ is the central component of amyloid plaques. It is made up by amyloid precursor protein(APP) which is cleaved by β,"-secretases. APP-a larger transmembrane protein, the extracellular fragment cleaved by #-secretase or β-secretase(BACE1) would produce N-terminal soluble APP fragments, named APPs# and APPsβ respectively. The residue in the cell membrane of C-terminal fragment of APP is called CTF # and CTF β, they are hydrolyzed by PS1, and then Aβ is formed.There are several forms of the Aβ protein formation in AD brains. Such as: soluble Aβ monomers, Aβ oligomers, and insoluble Aβ fibrils. All of the forms are toxic, which can lead to nerve cell degeneration and apotosis. But which form associates more closely with the pathogenesis of AD are unknown. People had regarded Aβ protein in the extracellular as the AD marker for the past a long time, it was also considered to start the degeneration and apoptosis of nerve cell. But recent researches suggest that the Aβ monomers and oligomers have stronger neurotoxicity compared with Aβ fibrils. It is thought that the Aβ oligomers are closely related to the pathogenesis and progression of AD. Recent studies have also highlighted the role of Aβ oligomers in synaptic impairment, suggesting that destroy the integrity of brain functions. And formation of amyloid plaques that develop in the later age appear to be rather late enent. Aβ peptides spontaneously aggregate into soluble oligomers and coalesce to form fibrils insoluble beta-sheet conformation and are eventually deposited in diffuse senile plaques. Aβ1-42 oligomers are produced by cooperative activities of both neurons and its associated astrocytes. It has been observed that Aβ 1-42 oligomers induce oxidative damage, promote Tau hyperphosphorylation, results in toxic effects on synapses and mitochondria. It has also been reported that Aβ1-42 oligomers possesses an increased capability for damaging cholesterol rich membranes. Such as those found in oligodendroglia and myelin. It is regarded that Aβ1-42 oligomers play a dominant role in AD,the major developing is targeted to Aβ1-42 oligomers based immunotherapy, which is a major key to unlock this disease in the furture.The phage display technique is a powerful tool for selection of various biological agents. In the phage display method, foreign peptides are expressed on the surface of a bacteriophage. Phage display technology has been used for selection and identification of human therapeutic antibodies, and it is estimated that nearly 30 % of human therapeutic antibodies that are under clinical trial have been developed by this method. In addition to the therapeutic values, phage display technique is frequently used for identification and confirmation purposes in research fields. Presentation of an antibody or foreign peptide on the surface of the phage is usually achieved by genetic fusion of nucleic acid sequence of the desire peptide to the sequence of one of the bacteriophages coat proteins. In phage display technique, the bacteriophage is used as an expression system that carries within the nucleotide sequence of a peptide expressed on its surface and in addition. Screening for antibodies from a phage display library is a multiple step process and after each step, antibodies that show highest affinity are chosen for amplification and the next round of panning. This can be achieved by increasing the washing solution stringency in sequential rounds, so while tight binders are retained, phages with lower affinity are removed. After these panning processes only antibodies with highest affinity toward a selected target are remained. These selection steps allow fast and easy isolation of monoclonal antibodies. Specific antibodies can be isolated based on distinctive characteristics from a library consisting of millions of members. These features made phage display technology preferred method for antibody selection and engineering.Phage antibody library technology is an important source of the production of specific antibodies, which provides the basis and guarantee for the monoclonal antibody treatment on AD.First part, Objective: To construct a library of human AD specific single chain Fv(scFv) antibodies via phage display. Methods: We collected 20 AD patients’ peripheral blood and isolated total RNA from lymphocyte. Using RT-PCR, we amplified the variable heavy(VH) and variable light(VL) genes and obtained the scFv fragments, and the scFv fragments were cloned into the vector pCANTAB5 E and electroporated into competent Escherichia coli TG1 cells. Consequently, an scFv phage display library was attained. Results: Total RNA was extracted successfully. The variable regions of human antibodies was amplified by RT-PCR and connected with each other to form scFv. The phage antibody library contained 2.0$108 independent clones. Result of BstN I digestion showed that it had good diversity. Conclusion: The phage display library of human AD specific single chain Fv antibodies is constructed successfully. It establishes the foundation for screening the Aβ1-42 oligomers antibodies for further therapeutic research on Alzheimer’s disease.Second part, Objective: Screened anti-Aβ1-42 antibodies from human AD specific single chain Fv(scFv) antibodies via phage display library. Methods: We put colony units of scFv phage antibody library in 96 wells coated with synthetic Aβ1-42 oligomers. Recombinant phages specific for Aβ1-42 were enriched after four rounds of biopanning and ELISA for positive clones(referred to as 11A5) was performed. 11A5 was used to infect E.coliHB2151 to express soluble scFv and was evaluated by SDS-PAGE and Western blot. Results: The percentage of the phage antibody was increased. Most of the cloned recombinant phage displayed higher antigen binding activity. Conclusion: The soluble scFv antibodies are expressed successfully. We chose the highest activity of 11A5 for the next research.Third part, Objective: To observe anti-Aβ1-42 antibody 11A5 effect in APP-PS1 transgenic mice. Methods: we mixed 11A5 and the monoclonal antibody(6E10) with Aβ1-42 oligomers respectively, made intracerebroventricular injections on Sprague-Dawley(SD) rats to compare with those only injected with Aβ1-42 by Morris water maze after 30 days. We injected 11A5 or 6E10 into APP-PS1 transgenic mice every 2-week, assessed the secretion of Aβ in peripheral blood and the levels of Aβ in brains at 1, 2, 3 month. Meantime, we evaluated the behavior changes by Morris water maze. Results: The behavioral changes of SD rats in Aβ1-42 mixed antibodies groups showed no statistical difference compared with sham group. In the observation of APP-PS1 mice, Aβ levels in peripheral blood of antibodies treatment groups were highest but the Aβ levels in brains of antibodies treatment groups were lowest, compared with control group,especiately at 1 month. Antibodies treatment groups’ behavioral abilities were better than control group. Conclusion: 11A5 can combine with Aβ1-42 and make Aβ1-42 deactivated. And the anti-Aβ1-42 antibodies(11A5) therapy is effective in decreasing cerebral Aβ burden in AD mouse models.We successfully screened an anti-Aβ1-42 oligomers antibody(11A5) from a human scFv antibodies phage display library that we developed in-house. We also demonstrated the ability of 11A5 to combine with and deactivate Aβ1-42. Moreover, therapy with 11A5 effectively improved the behavioral abilities of APP/PS1 transgenic mice. Anti-Aβ1-42 antibody(11A5) can not only combine with and deactivate the Aβ1-42 peptide directly but also initiate Aβ efflux or clearance from the brain. These results suggest that humanized anti-Aβ antibodies therapy may have a positive effect in treating AD. |
PDF Full Text Request