Figure 1: Amplification of the Nsp2-GFP fragment by overlap PCR and the construction of PRRSV infectious clone pFL12-GFP. (a) Inserting site of gfp gene between proline and glycine of Nsp2 encoding region. (b) Amplifying strategy of Nsp2-GFP fragment by overlap PCR. F1~F3 and R1~R3 present primers. (c) PCR product of Nsp2-GFP gene amplified by overlap PCR. M, DL 2000 DNA Marker; Lane 1, Nsp2-GFP gene fragment. (d) The sequences of Nsp2-gfp and gfp-Nsp2 fusion regions. (e) Structure of pFL12-GFP plasmid. The Nsp-GFP gene fragment was inserted into Nsp2 encoding region at Spe I and Xho I sites, in which the gfp gene was fused with Nsp2 at 5′ and 3′ terminus and controlled by T7 promoter. (f) Restriction analysis of the pFL12-GFP plasmid. M, DL 2000 DNA Marker; Lane 1, digested fragments of the pFL12-GFP plasmid by Spe I and Xho I.