Recombinant equine interferon-beta 1: purification and preliminary characterization.

The research involves extracting equine interferon-beta 1 (EqIFN-beta 1) from Escherichia coli, purifying it, and testing its antiviral properties on different cell types. The project also developed a rapid testing method for the presence of EqIFN-beta 1 using monoclonal antibodies.

Extraction and Purification of EqIFN-beta 1

• The study used a process known as sequential chromatography to extract and purify EqIFN-beta 1 from Escherichia coli, a bacterium commonly used in genetic engineering.
• Three types of chromatography were used: hydroxylapatite, anion, and cation-exchangers. This rigorous process ensured that the protein obtained was over 98% pure.
• The purity of the protein was confirmed using sodium dodecylsulfate gel electrophoresis, gel permeation HPLC, and reverse-phase HPLC, all of which are common laboratory methods to analyze protein purity.

Characterization of EqIFN-beta 1

• Amino-terminal sequencing was carried out and revealed all molecules had an additional amino-terminal methionine. This meant all proteins maintained their unique initial amino acid, methionine, even after the extraction process.
• Once purified, the EqIFN-beta 1 protein was then exposed to equine dermal fibroblasts that were challenged with vesicular stomatitis virus (VSV), a common test virus.
• This protein demonstrated substantial antiviral properties, reducing viral growth by a significant margin, suggesting it can be a potent antiviral agent

Specificity and Neutralization Studies

• The study also tested the antiviral properties of EqIFN-beta 1 on different cell lines from different species (human, murine, ovine and bovine), which revealed that the antiviral activity of this protein is specific to the equine cells.
• To further explore this specificity, researchers developed a series of monoclonal murine antibodies that neutralize the antiviral action of EqIFN-beta 1. This means they engineered antibodies that can block EqIFN-beta 1 from acting against viruses.
• These antibodies and other serum could not neutralise human interferon-beta suggesting that the equine and human versions of this protein have different structural or functional properties.

Rapid Identification Method

• One of the most significant developments from this research is the creation of a new method to quickly identify EqIFN-beta 1 through a one-step solid-phase enzyme immunoassay.
• This test uses two of the monoclonal antibodies developed in the study to detect the presence of EqIFN-beta 1, providing a rapid and straightforward screening method.