Isolation and characterization of single‐chain Fv genes encoding antibodies specific for Drosophila Poxn protein

FEBS Letters

De Baetselier, Patrick¹  Hamers, Raymond¹  De Silva, Kumudu S.K.¹  Hassanzadeh Gh, Gholamreza¹  Muyldermans, Serge¹  Dambly-Chaudière, Christine³  Ghysen, Alain²  Brys, Lea¹ 

[1]Department of Ultrastructure, Immunology and Parasitology, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium

[2]Laboratory of Neurobiology, Université Libre de Bruxelles, 67 rue des Chevaux, 1640 Rhode-St-Genèse, Belgium

[3]Laboratory of Genetics, Université Libre de Bruxelles, 67 rue des Chevaux, 1640 Rhode-St-Genèse, Belgium

关键词: Poxn;    Intrabody;    Single-chain variable fragment;    Drosophila;    ABTS;    2′;    2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium;    BCIP;    5-bromo-4-chloro-3-indolyl phosphate;    BSA;    bovine serum albumin;    cDNA;    complementary DNA;    CDR;    complementarity determining region;    EDTA;    ethylene diamine tetraacetic acid;    ELISA;    enzyme-linked immunosorbent assay;    globH5;    globular domain of histone H5;    HRP;    horseradish peroxidase;    IPTG;    isopropylthio-β-d-galactoside;    mAb;    monoclonal antibody;    NBT;    nitrotetrazolium chloride blue;    PBS;    phosphate-buffered saline;    PCR;    polymerase chain reaction;    PMSF;    phenylmethylsulfonyl fluoride;    scFv;    single chain variable fragment;    SDS-PAGE;    sodium dodecyl sulfate-polyacrylamide gel electrophoresis;    VH;    heavy chain variable domain;    VL;    light chain variable domain;   

DOI  :  10.1016/S0014-5793(98)01204-6

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The usefulness of intrabodies as specific inhibitors of gene function has been extensively demonstrated in cell culture assays. However, very few experiments have been conducted with intrabodies expressed in whole organisms. To evaluate the intrabody technology in Drosophila, we focused on poxn protein, since its effects can be easily studied. We purified the recombinant poxn protein. We next isolated three single-chain variable fragments (scFv) which specifically recognize poxn protein. Two scFvs, designated α-Poxn2 and α-Poxn4, react with both denatured and native Poxn with half maximal inhibition values of 100 nM and 40 nM, respectively. The α-Poxn5 scFv also recognizes denatured Poxn but either does not recognize native Poxn or its half maximal inhibition value for native Poxn is high.

【 授权许可】

Unknown