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image of Shepherin II Gene Synthesis and Peptide Characterization: E. coli Expression, Purification, and Antiviral Activity

Abstract

Introduction

The shepherin II peptide is characterized by a histidine/glycine-rich sequence. This study aimed to design, express recombinantly, and evaluate the antiviral activity of shepherin II against hepatitis A virus (HAV).

Methods

The shepherin II gene was reverse-translated, cloned into the pET-3a vector, and expressed in BL21 (DE3) pLysS cells induced with 2 mM IPTG. Purification was achieved cation exchange chromatography, and intact mass analysis using mass spectrometry was carried out. Cytotoxicity on normal Vero cells and antiviral activity on HAV were evaluated.

Results

The mass spectrometry confirmed a primary peptide fragment with a molecular weight of 3,421.30 Da (100% relative abundance). SDS-PAGE verified peptide expression. Cytotoxicity tests on Vero cells showed a CC of 219.26 ± 7.91 µg/ml. Antiviral assay revealed an EC of 113.92 ± 4.58 µg/ml against HAV, resulting in a selectivity index (SI) of 1.92. This SI indicates limited selectivity compared to the reference drug amantadine, which exhibited an EC of 5.67 ± 0.71 µg/ml and an SI of 53.41.

Discussion

The recombinant expression of shepherin II was successfully achieved and confirmed by mass spectrometry and SDS-PAGE. The peptide showed measurable antiviral activity against HAV.

Conclusion

This study demonstrated the feasibility of recombinant shepherin II production and assessed its antiviral activity. However, the limited selectivity index of shepherin II remains a challenge that needs to be addressed through molecular modification or alternative delivery strategies to improve its clinical potential.

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2025-10-23
2025-11-29

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Shepherin II Gene Synthesis and Peptide Characterization: E. coli Expression, Purification, and Antiviral Activity
Bentham Science Publishers ; https://doi.org/10.2174/0109298665413796251002111415
/content/journals/ppl/10.2174/0109298665413796251002111415
/content/journals/ppl/10.2174/0109298665413796251002111415
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  • Article Type:     Research Article
Keywords: Histidine-glycine rich peptide ; shepherin peptides ; HAV ; recombinant DNA technology ; antimicrobial peptide ; antiviral activity

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