The plasmids were used for overexpressing 6 × His-AACT recombinant protein, which the full open reading frame of EhAACT was subcloned into, were transformed into Rosetta cells. The cells were grown at 37 °C for more than 16 h, and then induced by the addition of isopropyl-d -thiogalactopyranoside. Cells were collected by centrifugation and then ultrasonic broken in ice bath until solution becomes clear. The supernatant obtained after centrifugation was collected, filtered and performed affinity chromatography. Recombinant AACT proteins were purified as His fusion proteins using a nickel-nitrilotriacetic acid agarose column according to the manufacturer’s instructions. Different concentrations of imidazole (50, 100, 200, 300 and 500 mmol/L) were used to elute, and the best concentration of imidazole elution was determined by SDS-PAGE. Then the purified EhAACT recombinant protein was obtained.
Protein concentrations were determined with SDS-PAGE, and then the gel pieces containing the recombinant proteins were extracted and injected directly into health rabbits. Antibodies from the rabbits were affinity purified using Cyanogen bromide-activated sepharose (Sigma-Aldrich, USA) conjugated with recombinant proteins and the specificity of antibodies was proved by the ELISA and immunoblot (WB).
Protein concentrations were determined with SDS-PAGE, and then the gel pieces containing the recombinant proteins were extracted and injected directly into health rabbits. Antibodies from the rabbits were affinity purified using Cyanogen bromide-activated sepharose (Sigma-Aldrich, USA) conjugated with recombinant proteins and the specificity of antibodies was proved by the ELISA and immunoblot (WB).