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

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Shepherin II peptide features a histidine/glycine-rich sequence.
• Hepatitis A virus (HAV) poses a significant public health concern.
• Developing novel antiviral agents is crucial for combating viral infections.

Purpose of the Study:

• To design and recombinantly express the shepherin II peptide.
• To evaluate the antiviral efficacy of shepherin II against HAV.
• To assess the safety and selectivity profile of shepherin II.

Main Methods:

• Shepherin II gene reverse-translation and cloning into pET-3a vector.
• Recombinant expression in E. coli BL21 (DE3) pLysS cells.
• Purification via cation exchange chromatography and mass spectrometry analysis.
• Cytotoxicity and antiviral assays on Vero cells and HAV.

Main Results:

• Successful recombinant expression and purification of shepherin II confirmed by mass spectrometry and SDS-PAGE.
• Shepherin II exhibited a CC50 of 219.26 ± 7.91 μg/ml on Vero cells.
• Antiviral activity against HAV showed an EC50 of 113.92 ± 4.58 μg/ml, yielding a selectivity index (SI) of 1.92.
• The SI of shepherin II was considerably lower than that of amantadine (SI = 53.41).

Conclusions:

• Recombinant production of shepherin II is feasible.
• Shepherin II demonstrates measurable antiviral activity against HAV.
• The limited SI of shepherin II necessitates further investigation for therapeutic development.