Baculovirus-mediated promoter analysis of orf427 compared with immediate-early gene ie1. A) Genomic organization of vAc-Proie1-EGFP and vAc-Pro427-EGFP. Pie1, promoter of ie1 gene; P427, promoter of orf427. Recombinant baculoviruses were constructed using the Bac-To-Bac system (Invitrogen). The EGFP-expressing cassettes were first cloned into the pFastBac1 shuttle vector at the indicated restriction sites and then integrated into the bacmid genome through site-specific transposition. B) Promoter activity of orf427 and ie1 gene in insect SF9 cells. Brightfield and EGFP fluorescence signals in SF9 cells infected with vAc-Proie1-EGFP and vAc-Pro427-EGFP at m.o.i of 10, respectively. C) Promoter activity of orf427 and ie1 gene in primary shrimp cells. Brightfield and EGFP fluorescence signals in primary shrimp cells transduced with vAc-Proie1-EGFP and vAc-Pro427-EGFP at m.o.i of 100, respectively. D) Western blot assay to confirm the expression of GFP in virus-infected or transducted cells. 1. Protein marker; 2. vAc-Proie1-EGFP infected SF9 cells; 3. vAc-Pro427-EGFP infected SF9 cells; 4. vAc-Proie1-EGFP transduced shrimp primary cells; 5. vAc-Pro427-EGFP transduced primary shrimp cells.