Abstract
Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1) and plectasin (PLSN) were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN) as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro) with half-maximal inhibitory concentration (IC50) 0.5±0.1 μM. The real-time proliferation assay (RTCA) and the end-point proliferation assay (MTT assay) showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities.
Highlights
Dengue virus is a member of the Flaviviridae family, transmitted by the mosquito vector Aedes aegypti [1]
PG1 peptide was joined to the N-terminal portion of MAP30 by using a 10-amino-acid linker and PLSN peptide was joined to the C-terminal of MAP30 using a similar linker
The results showed significant (p,0.001) reduction in the DENV2 load that was expressed as plaque forming units per ml (p.f.u./ml) after treatment with all the peptides compared to untreated cells (Fig. 5A and 5B)
Summary
Dengue virus is a member of the Flaviviridae family, transmitted by the mosquito vector Aedes aegypti [1]. Dengue virus infects 50– 100 million people worldwide each year and causes various clinical symptoms such as dengue fever (DF) that may later develop to severe dengue haemorrhagic fever (DHF), and dengue shock syndrome (DSS) [2,3,4]. There are approximately 0.5 million cases of DHF and DSS that lead to more than 20,000 deaths worldwide [5].The severe syndromes caused by dengue infection translate to serious economic burden in more than 100 tropical and sub-tropical countries [5]. One of the main hindrances for successful production of these peptides using chemical synthesis are the high costs involved, and is currently deemed uneconomic especially to achieve the required volumes for epidemic response. Production of these peptides in recombinant form would be cost-effective if a suitable expression system is establish to be scalable and well suited for mass production of bioactive peptides
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