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Convenient plasmid vectors for construction of chimeric mouse/human antibodies
Author(s) -
Kameyama Koh-zoh,
Imai Kenji,
Itoh Toshihiro,
Taniguchi Masaru,
Miura Keiji,
Kurosawa Yoshikazu
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)80550-2
Subject(s) - ecori , hindiii , microbiology and biotechnology , plasmid , gene , kappa , biology , chimeric gene , recombinant dna , antibody , chemistry , genetics , gene expression , linguistics , philosophy
Chimeric antibodies composed of mouse‐derived variable regions and human‐derived constant regions have been developed for clinical use. However, construction of chimeric mouse/human genes in expression vectors is time‐consuming work. In this study, we developed convenient vectors for construction of chimeric mouse/human antibodies. The protocols are as follows: In mouse hybridomas and B cells, most active V H and V ϰ genes can be identified as rearranged bands by Southern hybridization of Eco RI‐ and Hin dIII‐digested DNAs with J H and J ϰ probes, respectively, and such fragments can be isolated in λ‐ Eco RI and λ‐ Hin dIII vectors, respectively. We constructed two plasmids: pSV2‐HG1gpt contains human C γ1 and Ecogpt genes, and only one Eco RI site upstream of the C γ1 gene; pSV2‐HC ϰ neo contains human C ϰ and neo genes, and only one Hin dIII site upstream of the C ϰ gene. An isolated Eco RI fragment containing a V H D H J H gene and a Hin dIII fragment containing a V ϰ J ϰ gene are inserted into pSV2‐HG1gpt and pSV2‐HC ϰ neo, respectively. Both resulting plasmid DNAs are co‐transfected into SP2/0 cell, a non‐Ig‐secreting mouse myeloma. Transformants are selected by both mycophenolic acid and G418. With this procedure, it takes only 2 months to obtain chimeric antibodies.