Bl21 de3

The bacterial strains used in this study were Chromobacterium violaceum 026 (CV026), Bacillus sp. strain QSI-1 [17 (link)], Aeromonas hydrophila YJ-1 [31 ] and E. coli strain BL21(DE3) (Promega, USA). Bacillus sp. strain QSI-1 and E. coli strain BL21(DE3) were cultured in Luria-Bertani (LB) broth medium (Beijing Aoboxing Bio-Tech CO., Ltd., China) at 150 rpm and 37 °C. C. violaceum 026 was cultured overnight in LB broth, at 150 rpm and 28 °C. A. hydrophila YJ-1 was cultured in LB broth, at 150 rpm and 30 °C for 18–24 h, and bacterial cells were washed with sterile physiological saline three times, and suspended in physiological saline as injection preparation. For long-term storage, bacterial strains were preserved at −70 °C in LB containing 15% (v/v⁻¹) glycerol.

The EtMIC2 gene was then cloned into the pET32a(+) vector (Novagen, Merck KGaA, Darmstadt, Germany) using the EcoRI and HindIII sites. The recombinant plasmid was confirmed by DNA sequencing and transformed into the E. coli BL21 (DE3) (Promega) expression strain. The expression of rEtMIC2 in E. coli was induced using 0.8 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 8 h. The induced bacterial cells were collected by centrifugation and sonicated on ice. The supernatant was collected, and the recombinant protein was purified using a His Bind Purification kit (Novagen). The purified protein was analyzed with 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and its concentration determined using a BCA protein assay kit (Novagen). The purified protein was then stored in aliquots at −20 °C until further use [7 ].