Generation of the Fluorescent HPV16 E7 Protein for Detection of Delivery in vitro

Background: Immunotherapies targeting the human papillomavirus (HPV) oncogenic proteins, E6 and E7, are effective to treat HPV-associated cervical malignancies. Objective: The main objective of this study was to generate the fluorescent HPV16 E7 protein for detection of delivery in vitro. Methods: Two types of the fusion E7-GFP proteins (i.e., with or without linker) were expressed in different E. coli strains. Then, the efficiency of GFP and E7-GFP transfection was compared with FITC-antibody protein control using TurboFect reagent in the HEK-293T cell line. Results: Our data indicated that both E7-GFP fusion proteins were efficiently produced in M15 E. coli strain, but not in BL21 or Rosetta strains. The E7-GFP fusion showed a clear band of ~ 50 kDa in SDS-PAGE. Moreover, the E7-GFP protein maintained the fluorescent properties only when there was a distance between E7 and GFP genes, suggesting a promising potential to use GFP fusion protein in generating soluble form of protein. This fluorescent property was stable and could be detected in vitro. Moreover, the HEK-293T cells transfected by GFP/TurboFect and E7- GFP/TurboFect complexes demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the GFP fluorescence was stable even at 24 h post-transfection. Conclusion: Briefly, the E7-GFP fusion protein with linker can be useful for the development of protein vaccines against HPV16 infections and detection in vivo.