title: 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.
authors.affiliation: Laboratorio de Virología, Centro de Investigación Biomédica de Oriente, Instituto Mexicano del Seguro Social, 74360 Metepec Puebla, Mexico. jreyesleyva@correo.unam.mx
authors.forename: Gerardo
authors.initials: G
authors.lastname: Santos-López
authors.forename: Esmeralda
authors.initials: E
authors.lastname: Flores
authors.forename: Rocío
authors.initials: R
authors.lastname: Baños
authors.forename: Irma
authors.initials: I
authors.lastname: Herrera-Camacho
authors.forename: Julio
authors.initials: J
authors.lastname: Reyes-Leyva
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D015707:HN Protein / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D009438:Neuraminic Acids / Q000737:chemistry / Q000378:metabolism; D019213:Rubulavirus / Q000737:chemistry* / Q000378:metabolism; D013552:Swine; D013696:Temperature
chemical_list: D015707:HN Protein; D009438:Neuraminic Acids