[Vector construction and expression of anti-Aβ human-mouse chimeric antibody against Alzheimer's disease].
OBJECTIVE
To construct and to express a human-mouse chimeric antibody against Aβ peptide involved in Alzheimer disease by genetic antibody engineering with reducing of its human anti-mouse antibody response.


METHODS
Total RNA was extracted from a murine hybridoma cell line that secreted anti-Aβ monoclonal antibody. The entire gene coding heavy and light chains were amplified using RT-PCR and analyzed by Genebank Blast. The chimeric antibody gene was acquired by variable region gene of the monoclonal antibody with constant region gene of human IgG, in which point mutations were incluced by recombinant PCR technology, respectively. The eukaryotic expression vectors established by cloning chimeric antibody genes of the heavy and light chains into 3.1 were co-transfected into COS-7 cells. The expressed products were analyzed using ELISA and immunohistochemistry subsequently.


RESULTS
Genebank Blast analysis showed that the entire cloned antibody genes were in accordance with the murine antibody genes. DNA sequencing confirmed that the expression vectors of chimeric antibody were constructed successfully after splicing the variable region and constant region sequences. By co-transfecting COS-7 cells, a chimeric antibody was produced and collected in the culture medium. The antibody was humanized and bound Aβ specifically by ELISA and immunohistochemistry evaluations.


CONCLUSIONS
Expression vector of chimeric antibody against Aβ was constructed successfully and expressed in the eukaryotic cells. It provides a solid base for developing diagnostic and therapeutic methods for Alzheimer's disease in clinic and paves a way for a further humanization in the future.
Department of Pathology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China.
De
D
Chang
Jian-hua
JH
Zhang
Xue-mei
XM
Zhao
Ping
P
Liang
D000544:Alzheimer Disease / Q000276:immunology / Q000378:metabolism*; D016229:Amyloid beta-Peptides / Q000276:immunology* / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D005822:Genetic Vectors; D006801:Humans; D006825:Hybridomas / Q000166:cytology / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000378:metabolism; D051379:Mice; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA; D014162:Transfection
D016229:Amyloid beta-Peptides; D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D011993:Recombinant Fusion Proteins