Virus and Cells
The SADS-CoV GDS04 strain was isolated from piglets with severe diarrhea in our laboratory (Xu et al., 2019), and the virus was propagated in DMEM supplemented with 10 μg/mL trypsin (Gibco, USA) and cultured under the conditions described below.
Human embryo kidney (HEK) 293 T (ATCC CRL-1573) and Vero cells were obtained from ATCC (ATCC number: CCL-81) (USA). IPI-FX cells (X. Wang et al., 2019) were kindly provided by Professor Shaobo Xiao (Huazhong Agricultural University, China). All cells cultured in Dulbecco’s modified eagle medium (DMEM) (Hyclone, USA), supplemented with 10% fetal bovineserum (FBS) (BOVOGEN, Australia). Human embryo kidney (HEK) 293 F and insect Sf9 cells were obtained from Wen’ s Foodstuffs Group Co., Ltd (Guangdong, China), grown in Celkey CD HEK293 202 medium (IOSCIENCES, China) and SF-FSM serum-free medium (SuZhou World-medium Biotechnology Co. Ltd, China), respectively. All mediums supplemented with 100 U/mL penicillin, and 100 U/mL streptomycin. All cells were incubated in 37℃ with 5% CO2 incubator.
Construction of Plasmids and recombinant baculovirus generation
The region encoding the S protein of SADS-CoV strain GDS04 (GenBank no. MG742313.1) was analyzed by using Uniprot software (https://www.uniprot.org/uniprot/A0A3G1II32). The domain containing amino acids (aa) 19-1068 of S protein, which remove signal peptides, transmembrane regions and cytoplasmic regions, was 3’ terminally fused with a cysteine protease cleavage aequence (TEV, encoding ENLYFQG) and flexible peptide (FP, encoding GGSGG), followed by Fc domain of human IgG1 (GenBank no. MH975516.1), synthesized by Suzhou GENEWIZ Biotechnology Co. Ltd (China), and then cloned into theNhe I-Xho I restriction sites of the eukaryotic expression vector pCMV (Wen’ s Foodstuffs Group Co., Ltd, China) to yield the recombinant plasmid pCMV-S-Fc (Fig 1).
The plasmid pCMV-S-Fc were then chemically transformed into competent DH10BacTM Escherichia coli (E. coli ) cells (Invitrogen, USA), and the clones were confirmed with sequencing analysis by Suzhou GENEWIZ Biotechnology Co. Ltd (China). Recombinant baculovirusgeneration was performed as previously described with some modifications (Yin et al., 2016). Briefly, the obtained recombinant bacmids were transfected into Sf9 cells using Cellfection® Ⅱ Reagent (Gibco, USA) according to the manufacturer’s instruction, and incubated for 7 days. The target recombinant baculoviruses were then harvested from the supernatant.