Recombinant PvRAMA Protein Expression

Recombinant PvRAMA protein was expressed as described in a previous study using the wheat-germ cell-free (WGCF) technique [28 (link), 29 (link)]. Briefly, the C-terminal of pvrama was amplified by PCR and cloned to the pEU-His vector, then expressed by the WGCF system. Recombinant PvRAMA protein was purified using a nickel-charged nitrilotriacetic acid (NTA) agarose column (Qiagen, Hilden, Germany). PvRAMA proteins were denatured with reducing sample buffer, separated in a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and then stained with Coomassie brilliant blue. For immunoblotting, horseradish peroxide (HRP)-conjugated anti-His antibody (ABclonal, Wuhan, China) was used to detect PvRAMA (Additional file 1: Figure S1).