Pcaggs expression vector

Human codon-optimized coding sequences of MERS-CoV nsp3-6 were designed using GeneArt, ordered from Thermo Fisher in four fragments, and subsequently assembled in low-copy-number vector pACNR1180 (64 (link)) using conventional cloning. The precise parts of MERS-CoV pp1a used for polyprotein constructs are outlined in Table S1 in the supplemental material. The nsp4 construct included the 21 C-terminal aa of nsp3 to prevent the N-terminal hydrophobic region of nsp4 from acting as a signal sequence, which could result in improper membrane insertion. In all constructs with a C-terminal myc or V5 tag, the C-terminal glutamine of the viral sequence was omitted to prevent the removal of the tag by M^pro. The SARS-CoV nsp3 gene (Frankfurt 1 strain, pp1a amino acids 819 to 2740) was synthesized by Bio Basic Inc. (Ontario, Canada). Coding sequences were transferred to the pCAGGS expression vector (Addgene) for expression. pCAGGS-SARS-nsp4 was described previously (42 (link)). 293T cells were transfected using Lipofectamine 2000 (Thermo Fisher) according to the manufacturer’s instructions. HuH-7 cells were transfected using a Nucleofector 2b device (Lonza) with Nucleofector kit T (Lonza) in 6 × 10⁶ cells and 12 µg of plasmid DNA per transfection. Cotransfections were carried out with equimolar amounts of plasmids.

The optimized sequences of mpox virus H3, M1, A29, A35, B6, E8, and VACV H3, L1, A27, D8, B5, A33 fused with N-terminal native signal peptides and C-terminal 8× His tag respectively, then cloned into the pCAGGS expression vector (Addgene). The plasmids were transiently transfected into HEK293T cells. After 5 days, the supernatant was collected and purified by a HisTrap HP 5 ml column (GE Healthcare). Each sample was purified via size-exclusion chromatography with a Superdex 75 column (GE Healthcare) in PBS. To prepare antibodies against the antigens of mpox, the variable region links to the sequence encoding the constant region of mouse IgG2a, resulting in a full-length H chain and L chain plasmid. MAbs expressed by co-transfection of HEK293T cells. The antibodies were isolated by Hitrap Protein A 5 mL column (GE Healthcare) and purified by Superdex 200 column (GE Healthcare) in PBS.

The coding sequences of SARS-CoV-RBD (residues 306–527, accession number: NC_004718), SARS-CoV-2-RBD (residues 319–541, accession number EPI_ISL_402119), hACE2 (residues 19–615, accession number BAJ21180), and hACE2 variants (S19P, I21T, K26R, N33D, and D38E) fused with N-terminal native signal peptides and C-terminal 6× His tag were, respectively, cloned into the pCAGGS expression vector (Addgene) using the EcoRI and XhoI restriction sites. The signal peptides and variable regions of h11B11 were synthesized (GenScript) and fused with the coding sequences for the human IgG₄ and kappa light chain constant region into the pCAGGS vectors. The pEGFP-N1-hACE2 plasmid was constructed by cloning the coding region of hACE2 into pEGFP-N1 using restriction enzymes XhoI and SmaI. To express minimal glycosylated ACE2, a coding sequence of residues 19–615 was synthesized (GenScript) and cloned into pFastBac1 vector (Invitrogen), with an N-terminal gp67 signal peptide and a C-terminal 6×His tag.