The tag-less CFA/I/II/IV MEFA chimeric gene was PCR amplified from 6xHis-tagged CFA/I/II/IV MEFA plasmid DNA  with primers CFANcoI-F (5′-catgccatggaaatggctagcgcagtagaggat-‘3; NcoI site underlined) and T7-R (5′-tgctagttattggtcaggggt-‘3). PCR products were purified, digested with NcoI and EagI restriction enzymes (New England BioLabs, Ipswich, MA), and ligated into expression vector pET28α (Novagen, Madison, WI). The NcoI restriction site is located at the upstream of the 6xHis-tag region in vector pET28α, thus the new CFA/I/II/IV MEFA chimeric gene should not carry his-tag nucleotides. The cloned tag-less MEFA gene was DNA sequenced.