Efficient amplification and direct sequencing of mouse variable regions from any immunoglobulin gene family

FEBS Letters

Devauchelle, Gérard²  Chardès, Thierry²  Piechaczyk, Martine³  Ferrières, Gaelle¹  Villard, Sylvie¹  Cerutti, Martine²  Pau, Bernard¹ 

[1] CNRS UMR 9921, Faculté de Pharmacie, 15 Avenue Charles Flahault, 34060 Montpellier, France;INRA-CNRS URA 2209, Laboratoire de Pathologie Comparée, 30380 Saint-Christol-Lez-Alès, France;CNRS UPR 1086, CRBM-Laboratoire Infections Rétrovirales et Signalisation Cellulaire, 34060 Montpellier, France

关键词: Immunoglobulin;    Signal sequence;    V gene;    Polymerase chain reaction;    Oligonucleotide primer;    mAb;    monoclonal antibody;    CDR;    complementarity-determining region;    PDP;    paratope-derived peptide;    PCR;    polymerase chain reaction;    FR;    framework region;    S;    signal sequence;    Ig;    immunoglobulin;    VH;    variable region of the heavy chain;    Vκ;    variable region of the kappa light chain;   

DOI  :  10.1016/S0014-5793(99)00649-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

We have designed two original sets of oligonucleotide primers hybridizing the relatively conserved motifs within the immunoglobulin signal sequences of each of the 15 heavy chain and 18 kappa light chain gene families. Comparison of these 5′ primers with the immunoglobulin signal sequences referenced in the Kabat database suggests that these oligonucleotide primers should hybridize with 89.4% of the 428 mouse heavy chain signal sequences and with 91.8% of the 320 kappa light chain signal sequences with no mismatch. Following PCR amplification using the designed primers and direct sequencing of the amplified products, we obtained full-length variable sequences belonging to major (VH1, VH2, VH3, Vκ1 and Vκ21) but also small-sized (VH9, VH14, Vκ2, Vκ9A/9B, Vκ12/13, Vκ23 and Vκ33/34) gene families, from nine murine monoclonal antibodies. This strategy could be a powerful tool for antibody sequence assessment whatever the V gene family before humanization of mouse monoclonal antibody or identification of paratope-derived peptides.

【 授权许可】

Unknown