Pet32a

The recognized recombinant pMD19-T/HcSTP-1 plasmid was digested with dual restriction enzymes BamH I and Xho I and ligated into prokaryotic expression vector pET32a (+) (Novagen, USA). Finally, the successful cloned STP-1 gene in a recombinant expression vector was sequenced to confirm its placement in the accurate reading frame. The recombinant plasmid pET32a (+)-HcSTP-1 was transferred into E. coli strain (BL21) and induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG; Sigma-Aldrich) after the OD₆₀₀ of the culture reached 0.6 at 37°C. The cell pellet after centrifugation was lysed using 10 µg/ml of lysozyme (Sigma-Aldrich) followed by sonication, and was resolved on 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purification of recombinant protein was carried out according to the manufacturer’s instructions of Ni²⁺-nitrilotriacetic acid (Ni-NTA) column (GE Healthcare, USA). The histidine-tagged protein (empty pET32a) used as control protein in multiple assays in this study was purified and expressed similar to the procedure described for rHcSTP-1 protein and determined at 12% SDS-PAGE after Coomassie blue staining and quantified by Bradford method (24 (link)).

The restriction digestion of the plasmid (pMD19-T/ Ts-MAPRC2) was carried out using the enzymes viz., EcoR I and Hind III was cloned into the prokaryotic expression vector pET-32a (+) (Novagen, Beijing, China). Recombinant plasmid (pET32a (+)/Ts-MAPRC2) was then processed into BL21 (DE3) and induced protein expression by 1 mM IPTG (Isopropyl-β-D-thiogalactopyranoside) (Sigma-Aldrich, Shanghai, China). Further, cells were harvested and lysed by lysozyme (10 μg/mL) (Sigma-Aldrich, Kenilworth, NJ, USA), and by sonication. The sonicated outputs were subsequently confirmed on the SDS-PAGE (12% w/v). Recombinant Ts-MAPRC2 (rTs-MAPRC2) protein was purified by the His-TrapTM FF column following the manufacturer’s instructions (GE Healthcare, Piscataway, NJ, USA), and protein concentration was calculated by Pierce-TM BCA-Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Empty pET32a (histidine-tagged) protein was purified and expressed using the same procedure mentioned above for Ts-MAPRC2 and utilized vector-protein as a negative control. Pictures of the SDS-PAGE carrying the rTs-MAPRC2 purified protein were taken. The stock of rTs-MAPRC2 protein was prepared and preserved at −80 °C until the next experiments.

Y. ruckeri YRWEL01, a fish pathogen isolated from dying channel catfish in Sichuan province of China, was cultured in Brain-Heart Infusion (BHI) medium at 28°C and stored at our laboratory (17 (link)). Escherichia coli strains DH5α and BL21 (DE3) competent cells (Takara; Dalian, China) served as cloning and protein expression host, respectively. Both strains were grown in Luria-Bertani medium containing 100 μg/ml of ampicillin (Amp) at 37°C. Plasmids pMD19-T (Takara) and pET32a (+) (Merck, Germany) served as cloning and expression vectors, respectively. Montanide™ ISA763 A VG (Seppic, France) was selected for use as an adjuvant for the experiment.