Isolation and characterisation of deoxynivalenol affinity binders from a phage display library based on single-domain camelid heavy chain antibodies (VHHs)

Abstract

Deoxynivalenol (DON), or vomitoxin, is a mycotoxin of the trichothecene group produced by Fusarium species. The present work described construction of a phage display library based on variable domain of heavy chain of HCAbs (VHHs) and panning for binders of DON. Two pair PCR primers designed based on the VHHs conserved domains were used for cloning coding sequences of complementary DNA (cDNA). After total RNA extraction, semi-nested PCR, subcloning and electro transformation, the primary library was generated containing 1.9×10⁷ independent clones. Phage display library was rescued by infection of the primary library with helper phage KM13, and the titre of the library was 5.6×10¹² CFU/ml. Three clones with specific DON-binding affinity were obtained after three rounds of panning. Since the library was derived from non-immune animal, it has potential to be used for panning binders of other mycotoxins or low molecular weight haptens. The binders with specific affinity provided new recourses for developing detection methods or tools of mycotoxins in foodstuffs.

Keywords:

• deoxynivalenol
• mycotoxins
• polypeptide
• ELISA
• phage display

Acknowledgements

This work was supported by a grant of Innovation Fund for Technology Based Firms (09C26213604449) and by a grant of the Education Department of Jiangxi Province (GJJ09442). In addition, this work was supported by Natural Science Foundation of Jiangxi Province. Thanks to Dr. Cao Huabing of Jiangxi Agricultural University for the help with collecting blood samples and Dr. Ma Weijun for providing phagemid and helper phage.