Escherichia coli strain bl21

The cDNA for the mature peptide (amino acid residues 117-268) of rat IL-1β (DDBJ/EMBL/GenBank accession No. NM_031512) was amplified by PCR using the primers 5’-CCAGCATATGGTTCCCATTAGACAGCTGCACTG-3’ and 5’-CCGAATTCAGGAAGACACGGGTTCCATGG-3’. The amplified DNA fragment was digested with Nde I and EcoRI and cloned into the pCold III vector (Takara Bio Inc., Kusatsu, Shiga, Japan). The resultant construct and pLysSRARE2 plasmid (Novagen Inc., Madison, WI, USA) were introduced into Escherichia coli strain BL21 (Takara Bio Inc.). The transformed cells were grown and cold-shocked. The cells were harvested, lysed, and subjected to ammonium sulfate precipitation and dialysis, followed by ultrafiltration to purify the mature peptide, the activity of which was verified by comparison with that of recombinant rat IL-1β (PeproTec, Rocky Hill, NJ, USA).

Adult Japanese lampreys (L. japonicum), 25–30 cm long, were obtained from Harbin, China. The harvested lampreys were maintained at 16°C in a lab. Pelteobagrus fulvidraco were used to feed lamprey adults daily. Blood was drained from tail-severed adult lamprey and collected into anticoagulant tubes. Buffy coat leukocytes were collected with a peripheral blood lymphocyte separation kit (Solarbio, Beijing, China). Granulocytes, monocytes, and lymphocyte-like cells were sorted into 15 ml tubes by fluorescence-activated cell sorting (FACS).
Escherichia coli strain BL21 was purchased from TaKaRa. Herpes simplex virus-1 (HSV-1) was preserved in our lab. HEK293T cells and Vero cells were cultured in DMEM (Gibco, USA) with 10% (vol/vol) fetal bovine serum (Gibco, USA) at 37°C, 5% CO₂. Transient transfection was conducted with jetPRIME (Polyplus Transfection, France) according to the manufacturer’s instructions.