The Distinct Role of Small Heat Shock Protein 20 on HCV NS3 Expression in HEK-293T Cell Line.

ABSTRACT:

Background

Hepatitis C (HCV) is known as a serious blood-borne disease that infects millions of people globally. NS3 is a conserved non-structural sequence of hepatitis C virus which has a major role in activating specific CTL responses. As known, there is no effective vaccine against HCV infection, thus it is required to design a specific regimen of vaccination. Recently, the strong immunological properties of Heat shock proteins (Hsps) led to their use as immunomodulators and an antigen carrier for subunit vaccine candidates. In the current study, the role of Hsp20 was evaluated as a HCV NS3 gene carrier in mammalian cell line.

Methods

At first, the recombinant plasmids of pEGFP-Hsp20, pEGFP-NS3, and pEGFP-Hsp20-NS3 were constructed and their accuracy was confirmed by digestion and sequencing. Then, all recombinant plasmids were transfected into HEK293T cells by Lipofectamine and TurboFect gene delivery systems. Finally, the expression of proteins was assessed by fluorescent microscopy, western blotting, and flow cytometry.

Results

In western blotting, the 47, 59, and 79 kDa bands were detected for pEGFP-Hsp20, pEGFP-NS3, and pEGFP-Hsp20-NS3, respectively. The percentage of NS3-Hsp20-GFP protein expression was ∼67% by TurboFect and ∼50% by Lipofectamine indicating high potency of TurboFect delivery system. Furthermore, the expression of Hsp20 (∼83%) was higher than NS3 (∼58%) in the cells transfected by TurboFect using flow cytometry analysis. This result was confirmed in the expression of Hsp20-NS3 fusion (∼67%) in which Hsp20 increased the delivery of HCV NS3 in vitro. The same data were obtained by Lipofectamine transfection reagent.

Conclusion

Briefly, our data confirmed the role of Hsp20 as a suitable antigen carrier for DNA vaccine design.

SUBMITTER: Basirnejad M 

PROVIDER: S-EPMC6063998 | biostudies-literature |

REPOSITORIES: biostudies-literature