Construction of sub-genomic replicon of foot-and-mouth disease containing EGFP reporter gene

Abstract:

[Objective] To construct sub-genomic replicon system of foot-and-mouth disease containing EGFP reporter gene. [Methods] Based on the infectious clone of serotype O FMDV, the subgenomic replicon FMDV-EGFP was constructed by replacing Lb and P1 gene of FMDV with EGFP reporter gene using fusion PCR method. The stability of replicon was detected by a series of transformation and sequencing. The replicon linearized with Not I was transfected into BSR/T7 cells expressing T7 RNA polymerase using liposome mediation. EGFP expression of different time of transfected cells was examined using fluorescence microscopy, the ability of self-replication and expression of protein of replicon were detected by flow analysis, immunofluorescence assay, RT-PCR and Western blot. [Results] The replicon vector was stable by a serious of transformation and sequencing. After 3 h transfection, the EGFP fluorescence could be obviously observed under the fluorescence microscope, the level of EGFP protein was increased gradually as transfection time went on. The result of flow cytometry analysis showed that 6.0% transfected cells were able to generate fluorescence, indicating the effective expression of EGFP protein of replicon vector. In addition, the results of immunofluorescence, RT-PCR and Western blot assay demonstrated that the replicon could autonomously replicate and express non-structural protein of FMDV. [Conclusion] The construction of sub-genomic replicon of FMDV expressing EGFP reporter gene provides a foundation to study viral replication and translation mechanism and antiviral drugs.