Expression and purification of an anti-clenbuterol single chain Fv antibody in Escherichia coli.
Recombinant antibodies with desirable characteristics that can replace polyclonal or monoclonal antibodies are important for enzyme-linked immunosorbent assay (ELISA) of residues of clenbuterol (CBL), an illicit veterinary drug. Here, we report our work on expression and purification of a mouse-derived anti-CBL single chain Fv (scFv) antibody in Escherichia coli (E. coli). An expression plasmid pBV220-CBL was constructed and transformed into E. coli BL21 (DH3) strain cells. After induction by temperature, the 6x His-tagged anti-CBL scFv antibodies were expressed with the yield of 31%. The solubilized inclusion bodies were extracted, denatured and then purified by Ni-NTA column chromatography. The purified recombinant target protein was analyzed by high performance liquid chromatography, SDS-PAGE and Western blotting, respectively. The results showed the prepared anti-CBL scFv antibodies posed HRP-anti-His-tag antibody-recognized activity and their purity was up to 96%. Moreover, an indirect competitive ELISA based on the anti-CBL scFv antibodies revealed that the limit of detection for CBL was 0.5 ng/ml and the linear range was 1.5-10.6 ng/ml. Taken together, these findings suggest that the prepared recombinant antibody can be used for future immunoassay detection for CBL.
Wang
Hong
H
Key Laboratory of Food Quality and Safety of Guangdong Province/College of Food Science, South China Agricultural University, Guangzhou 510642, PR China.
Liu
Xixia
X
He
Yongsheng
Y
Dong
Jiexian
J
Sun
Yuanming
Y
Liang
Yan
Y
Yang
Jinyi
J
Lei
Hongtao
H
Shen
Yudong
Y
Xu
Xiaoyan
X
D000318:Adrenergic beta-Agonists / Q000032:analysis / Q000276:immunology*; D000818:Animals; D002976:Clenbuterol / Q000032:analysis / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D051379:Mice; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*
D000318:Adrenergic beta-Agonists; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D002976:Clenbuterol