The Transformation Of The Human Anti-a¦Â Mouse Monoclonal Antibody And Its Expression Identification

Alzheimer's disease(AD) is a degenerative disease of the central nervous system, progressive dysmnesia and recognization recession as main clinical features. It is neuropathologically characterized by the deposition of extracellularβ-amyloid (Aβ)-containing plaques, intracellular neurofibrillary tangles, reduced synaptic density and neuronal loss in selected brain areas. The aggregation of beta-amyloid (Aβ) peptides in the senile plaques is believed to contribute to neuronal dysfunction and death, and impairment of memory in AD patients. Research on preventing Aβassembly, reducing parenchymal plaque burden, has become a hopeful approach for AD therapy. In animal trials, direct administration of anti-Aβantibodies has proved successful in clearing amyloid from the brain and reversing memory deficits. However, anti-Aβantibodies used in animal trials are murine monoclonal antibody. For its application in the future, it is necessary to humanize the anti-Aβmurine monoclonal antibody.Based on the chimeric antibody constructed previously, we humanize the frame regions of the heavy chain and the light chain in variable regions using template-replacement method. We search the human FR gene sequence in GenBank firstly, which is homologous with that of the anti-Aβmurine monoclonal antibody properly. We choose GI₄7846573_EMB_CAE45451₁ as the FR gene sequence of the heavy chain and GI₁8025598_GB_AAK94808₁ as the FR gene sequence of the light chain respectively, then recombine the CDR gene sequence with FR respectively to construct the heavy and light entire antibodies. Eukaryotic expression vectors are got by inserting humanized antibody genes of the heavy and light chains into vectors. The vectors used to construct the heavy chain is pcDNA3.1(+) and the light chain's is pcDNA3.1(zeo). The entire heavy chain is about 1500bp, and the light chain is about 750bp.The PCR and sequencing results of the humanized anti-AβMcAb show The heavy chain of the recombinant antibody is about 1500bp and the light chain is about 750bp, which is in accordance with our anticipation. Then the eukaryotic expression vectors are co-transfected into CHO cells using liposome fusion method. We adopt the expression supernatant of the chimeric antibody as the positive control. The expressed product of the humanized antibody can bind Aβantigen specifically tested by ELISA, and its titer keeps the same as that of the chimeric antibody basically. The expression amount of the humanized antibody is 1.11mg/L and the value of the chimeric antibody is 1.38mg/L tested by ELISA. The expressed product can only be recognized by goat-anti-human antibody rather than goat-anti-mouse antibody. This result shows the humanized antibody was humanized, bearing none components of mouse IgG. The molecular weight of the humanized antibody is about 51ku (heavy chain) and 25ku (light chain) identified by SDS-PAGE and Western blot, which is accordance with the heavy and light chain. At the same time, there are results showing the expressed product can bind Aβantigen specifically tested by immunohistochemistry, which means the humanized antibody has biological activity.So far, the reports on the anti-AP antibody and the humanization of it are in phase of lab research. The report of successful clinical use of anti-Ap humanized antibodies hasn't been found. The humanized antibody of our study keeps the same as the chimeric antibody in biological activity basically, this provides a possibility for its application for Alzheimer's disease in the future.