Abstract:[Objective] This study prepared full-length and fragment antibodies of bovine immunoglobulin G (IgG) with a mammalian cell recombinant expression system and screened high-affinity nanobodies that specifically recognize bovine IgG based on phage display technology, aiming to provide new antibody tools for the detection and functional study of bovine IgG. [Method] By retrieving the bovine IgG gene sequences, the CH fragment, Fc fragment, CH3 fragment, and CL fragment of bovine IgG were cloned into the pTT5 eukaryotic expression vector and efficiently expressed in Expi293F cells. With natural bovine serum and recombinant Fc fragment as coating antigens, three rounds of biopanning were performed to screen for the nanobodies that specifically bound bovine IgG from a phage-displayed nanobody natural library. Positive clones were induced for expression in Escherichia coli Rosetta, and their specificity to IgG of other species as well as binding sites were determined. [Result] The expression vectors for the CH fragment, Fc fragment, CH3 fragment, and CL fragment of bovine IgG were constructed, and efficient recombinant expression of full-length bovine IgG as well as heavy and light chain fragments was achieved in Expi293F cells. The specific nanobodies recognizing bovine IgG were successfully screened from a phage-displayed natural nanobody library through biopanning with natural bovine serum as the target and prepared. [Conclusion] On the basis of phage display technology, two nanobodies with high specificity targeting bovine IgG were screened through a three-round biopanning strategy, and their recombinant expression and activity verification were successfully completed. This can provide a reference for subsequent adulteration detection in milk or beef products.
