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

Author(s): Sepideh Shahbazi, Azam Bolhassani*, Arash Arashkia, Esmaeil Sadroddiny*

Journal Name: Protein & Peptide Letters

Volume 25 , Issue 3 , 2018


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Abstract:

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.

Keywords: Human papillomavirus, E7, GFP reporter gene, prokaryotic expression system, in vitro protein delivery, oncogenic proteins.

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Article Details

VOLUME: 25
ISSUE: 3
Year: 2018
Published on: 15 January, 2018
Page: [244 - 252]
Pages: 9
DOI: 10.2174/0929866525666180115123620
Price: $65

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