Cloning of scFv gene and construction of pcDNA3.4-scFv expression vector

Amplification of the antibody variable region genes (VH and VL) was performed by nested PCR using primers for bovine IgG gamma and lambda chains (Ig γ and Ig λ; S1 Table)[20]. Because the λ chain accounts for 95% of the bovine light chains, only the λ light chain was amplified by PCR. The final PCR products of paired VH and VL were Sanger sequenced. Single-chain fragment variable (scFv) was designed by splicing the VH and VL genes using a flexible linker (GGGGSGGGGSGGGGS). The N- and C-termini of the scFv included a signal peptide (MNPLWTLLFVLSAPRGVLS) of bovine Ig γ and a His tag, respectively. The scFv genes were synthesized by GenScript Inc. (www.genscript.com) with codon optimization and were expressed in CHO cells, followed by cloning into the pcDNA3.4 vector. The cloned scFv-expressing plasmids were amplified in E. coli and extracted and purified using an Endo Free Maxi plasmid kit (TIANGEN, China).