| Alzheimer disease (AD) is a neurodegenerative disorder that results in memory deficits and cognitive decline. It is the leading cause of dementia. The actual mechanisms that contribute to the pathogenesis of AD are not known. The incidence of Alzheimer's disease increases over ages, but we can currently neither prevent nor cure it. However, compelling genetic and biochemical evidence suggests that accumulation of amyloid-β protein plays a central role. Animal studies have been very encouraging in that both active and passive immunization of transgenic mice can reduce amyloid load and reverse memory deficits found in these mice. Thus, Antibodies against Aβ is a potential therapeutic strategies for the treatment of AD.Now we have developed two cell line of fusion antibody against Aβ, using recombinant DNA technology, which is expressed in yeast system。 This antibody is composed by two parts. One part is designed to recongnize Aβ fibrils and conjugate it. The other comes from human IgG Fc segment. The fusion antibody acts as natural antibodies against amyloid-β peptide triggered microglialcell to clear plaques through Fc receptor-mediated phagocytosis. And because the Fc fragment is humanized, there won't be intensity inflammatory reactions and cerebral hemorrhaging associated with the treatments. It will have a large potential to the study of AD. To test the biological activity of the antibody in vivo, we need to expressed antibody with mouse Fc to see the curative effect in AD mouse model (APP695).The expression vector pAO815AFc containing single copy of wanted gene was constructed in previous work. The vector can be linearized by enzyme and transformed into Pichia pastoris strain GS115. we selected 18 Pichia recombinants, and using SDS-PAGE to test the final product. Both of the fusion antibodys' MW is 30KD. We have got 14 colonies of A1Fc and 16 colonies of A2Fc, they show good genetic stability during growth even under non-selective conditions. To increase the yield of fusion antibody and minimize the proteolytic process of fusion antibody secreted in the culture medium, the conditions of expressing and culturing were optimized and the culture of expression strain was scaled up to 1 liters, and fusion antibody is 90% account for total amount of protein in... |
