Convenient plasmid vectors for construction of chimeric mouse/human antibodies

FEBS Letters

Imai, Kenji²  Kameyama, Koh-zoh¹  Itoh, Toshihiro²  Miura, Keiji¹  Taniguchi, Masaru²  Kurosawa, Yoshikazu¹ 

[1] Institute for Comprehensive Medical Science, Fujita-Gakuen Health University, Toyoake, Aichi 470-11 Japan;Department of Immunology, Chiba University School of Medicine, Chiba 280, Japan

关键词: DNA;    recombinant;    Chimeric antibody;    Anti-phosphorylcholine;    Ab;    antibody;    Ag;    antigen;    C;    constant;    D;    diversity;    ELISA;    enzyme-linked immunosorbent assay;    H;    heavy;    Ig;    immunoglobulin;    J;    joining;    L;    light;    PAB;    p-azobenzenarsonate;    PAGE;    polyacrylamide gel electrophoresis;    PBS;    phosphate-buffered saline;    PC;    phosphorylcholine;    V;    variable;   

DOI  :  10.1016/0014-5793(89)80550-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 EcoRI- and HindIII-digested DNAs with J_H and J_ϰ probes, respectively, and such fragments can be isolated in λ-EcoRI and λ-HindIII vectors, respectively. We constructed two plasmids: pSV2-HG1gpt contains human C_γ1 and Ecogpt genes, and only one EcoRI site upstream of the C_γ1 gene; pSV2-HC_ϰneo contains human C_ϰ and neo genes, and only one HindIII site upstream of the C_ϰ gene. An isolated EcoRI fragment containing a V_HD_HJ_H gene and a HindIII fragment containing a V_ϰJ_ϰ gene are inserted into pSV2-HG1gpt and pSV2-HC_ϰneo, 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.

【 授权许可】

Unknown