Purification of the Porcine rubulavirus attachment protein by liquid isoelectric focusing

Abstract

Porcine rubulavirus (PoRV) is an emerging virus responsible for meningoencephalitis, respiratory distress, and reproductive alterations in pigs. The hemagglutinin-neuraminidase (HN) glycoprotein is the most exposed and antigenic of the virus proteins. HN plays central roles in PoRV infection; i.e., it recognizes sialic acid-containing cell receptors that mediate virus attachment and penetration; in addition, its neuraminidase (sialic acid hydrolysis) activity has been proposed to be a virulence factor. So, HN is an ideal target for therapeutic treatment and prevention of this viral infection. This work describes a simple, fast, and sensitive method to purify the active form of HN protein based on its isoelectric point. HN was purified at a pH of 4.4, at which a single protein band of 66 kDa was observed on SDS-PAGE. Pure HN showed a maximal enzymatic activity at pH 3.5 and 37 degrees C using bovine fetuin as substrate. However, it retains circa 80% of its activity at a wide temperature range from 30 to 55 degrees C. We also describe improvements of neuraminidase determination method, which permits analysis in a microplate spectrophotometer, thereby increasing the sensitivity and reducing the costs of valuable reagents and biological samples.