The construction of Flag-tagged expression plasmids (pCAGGS-Flag) encoding PRRSV nsp1α, nsp1β, nsp2, nsp3, nsp4, nsp5, nsp7, nsp8-9, nsp9, nsp10, nsp11, nsp12, N, M, GP2, GP3, GP4, and GP5 was performed in our laboratory. The genes of nsp and structural proteins were amplified from HP-PRRSV strain BB0907 by RT-PCR using the primers listed in Table S1 .
The truncated versions of nsp9 were subcloned from pCAGGS-nsp9 (including the Flag tag at the N terminus) and designated as pCAGGS-nsp9 (1–1377), pCAGGS-nsp9 (1–630), pCAGGS-nsp9 (631–1929), pCAGGS-nsp9 (1378–1929), pCAGGS-nsp9 (631–1377), pCAGGS-nsp9 (1–1800), pCAGGS-nsp9 (1–1650), and pCAGGS-nsp9 (1–1500). Alanine substitution mutations in the nsp9 genes were generated using PCR Phanta Super-Fidelity DNA Polymerase (Vazyme). All the constructs were confirmed by DNA sequencing, and the primers used for plasmid construction are presented inTable S2 .
The whole PCR fragments were ligated into the pCAGGS vector (Invitrogen), generating an N-terminal Flag tag fusion plasmid. The protein expression of each constructed plasmid was detected by Western blot, analysis using anti-Flag antibody (Flag).
The truncated versions of nsp9 were subcloned from pCAGGS-nsp9 (including the Flag tag at the N terminus) and designated as pCAGGS-nsp9 (1–1377), pCAGGS-nsp9 (1–630), pCAGGS-nsp9 (631–1929), pCAGGS-nsp9 (1378–1929), pCAGGS-nsp9 (631–1377), pCAGGS-nsp9 (1–1800), pCAGGS-nsp9 (1–1650), and pCAGGS-nsp9 (1–1500). Alanine substitution mutations in the nsp9 genes were generated using PCR Phanta Super-Fidelity DNA Polymerase (Vazyme). All the constructs were confirmed by DNA sequencing, and the primers used for plasmid construction are presented in
The whole PCR fragments were ligated into the pCAGGS vector (Invitrogen), generating an N-terminal Flag tag fusion plasmid. The protein expression of each constructed plasmid was detected by Western blot, analysis using anti-Flag antibody (Flag).