The TcMyD88 ORF was amplified from T. crocea cDNA by PCR using specific primers designed by CE Design V1.03. The target PCR products were subcloned into eukaryotic expression vectors, pcDNA3.1-HIS (Promega, USA) and pEGFP-N1 (Promega, USA), by homologous recombination using a Vazyme™ one step cloning kit. The constructed recombinant plasmids were digested with enzymes, and the inserted fragments of each clone were sequenced. All of the plasmids used for transfection were extracted from overnight bacterial cultures using a HiPure Plasmid EF Micro kit (Magen) according to the manufacturer’s protocol.
HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 mg/L streptomycin and 105 U/L penicillin, Gibco) at 37 °C with 5% CO2, and they were subcultured every three days. Transfections were performed using ViaFect reagent (Promega, USA) according to the manufacturer’s instructions.
HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 mg/L streptomycin and 105 U/L penicillin, Gibco) at 37 °C with 5% CO2, and they were subcultured every three days. Transfections were performed using ViaFect reagent (Promega, USA) according to the manufacturer’s instructions.