Synthesis of IL-2 and IL-4

Interleukins IL-2 and IL-4 of alpaca, necessary for in vitro immunization, were produced. In short, IL-2 and IL-4 coding sequences were retrieved from the GenBank ({"type":"entrez-nucleotide","attrs":{"text":"KM205215.1","term_id":"698372717","term_text":"KM205215.1"}}KM205215.1 and {"type":"entrez-nucleotide","attrs":{"text":"KM205216.1","term_id":"698372722","term_text":"KM205216.1"}}KM205216.1, http://www.ncbi.nlm.nih.gov/) and synthesized commercially (Invitrogen, Slovakia) with flanking sequences containing restriction sites for BamHI at 5′ and amber stop codon followed by SalI at 3′ end. DNA fragments were digested with BamHI and SalI (Thermo Fisher Scientific, Slovakia) and ligated into pQE-30-mCherry-GFP plasmid (Fig. 1, in-house modified vector pQE-30 UA, Qiagen). Please note that in this vector mCherry serves as stuffer sequence, which is cut out during the digestion of vector with restriction enzymes, whereas incorporation of amber stop codon at 3′ of IL gene ensures no fusion of GFP to ILs. Ligation mix was purified using NucleoSpin (Macherey–Nagel, Germany) and transformed into E. coli SG13009 for IL-2, and M15 strain for IL-4 (Qiagen, Germany). Transformants were selected from LB agar plates (lysogeny broth agar, 10 g/L tryptose, 10 g/L NaCl, 10 g/L yeast extract, 2% bacteriological agar) supplemented with 1% glucose (G), 5 µg/mL Kanamycin (K) and 5 µg/mL Carbenicillin (C). Presence of IL-2 or IL-4 encoding gene in transformants was confirmed by sequencing (vector specific primers UA Insertom F and R, presented in Table 1).

Vector map of pQE-30-mCherry-GFP plasmid (4880 bp). PT5 T5 promoter, lac O lac operator, RBS ribosome binding site, ATG Start codon, 6xHis His tag sequence, MCS I/MCS II multiple cloning sites; mCherry—red fluorescent protein that serve as stuffer, GFP, green flourescent protein; Stop codon; Col E1, Col E1 origin of replication; Ampicillin, ampicillin resistance gene. Note that incorporation of the stop codon at 3′ of insert produces recombinant protein without GFP fusion

Sequences of primers used in the PCR