Rational design and expression of a recombinant plant rhabdovirus glycoprotein for production of immunoreactive murine anti-sera

Abstract

Lettuce necrotic yellows virus (LNYV) is a plant rhabdovirus which has a type-1 transmembrane glycoprotein. Here, we describe the generation of murine anti-sera to the glycoprotein. Rational design, expression, and purification of recombinant glycoprotein, termed rLGe, was undertaken using SignalP4.1 and camSol servers to predict signal peptide cleavage and protein solubility. In order to successfully obtain expression in mammalian cells, LNYV glycoprotein native signal peptide was substituted with that of Rabies virus glycoprotein. In addition, rather than expression of the entire molecule, rLGe consisted of the LNYV glycoprotein ectodomain fused to two affinity tags to minimize the risk of protein aggregation, while facilitating detection and purification. rLGe was transiently expressed in mammalian cell culture, purified using affinity column chromatography, and used to immunize mice. Harvested anti-sera were immunoreactive and specific to the naturally occurring glycoprotein as confirmed by western blotting of plant leaf tissue infected with LNYV.