ABSTRACT
Recombinant expression of the norovirus capsid protein VP1 leads to self-assembly of non-infectious virus-like particles (VLPs), which are recognized as promising vaccine candidates against norovirus infections. To overcome the scalability issues connected to the ultracentrifugation-based purification strategies used in previous studies, an anion exchange-based purification method for norovirus VLPs was developed in this study. The method consists of precipitation by polyethylene glycol (PEG) and a single anion exchange chromatography step for purifying baculovirus-expressed GII.4 norovirus VLPs, which can be performed within one day. High product purity was obtained using chromatography. The purified material also contained fully assembled monodispersed VLPs, which were recognized by human sera containing polyclonal antibodies against norovirus GII.4.
Subject(s)
Chromatography, Ion Exchange/methods , Norovirus/genetics , Virology/methods , Virosomes/genetics , Virosomes/isolation & purification , Baculoviridae , Capsid Proteins/genetics , Capsid Proteins/metabolism , Fractional Precipitation , Genetic Vectors , Polyethylene Glycols/chemistryABSTRACT
Current therapeutic approaches to treat cancer are often hampered by the lack of specificity of the drugs used for therapy. Scavidin, a novel fusion protein expressed on cell membranes, could be utilized for targeting of therapeutic molecules. Scavidin exploits the high binding affinity between avidin and biotin and is capable of mediating endocytosis of a bound ligand. In the current study we evaluated the efficiency of biotinylated ultrasmall superparamagnetic iron oxide (USPIO) particles in Scavidin-expressing human umbilical vein endothelial cell (HUVEC) cultures in vitro as a novel receptor-targeted magnetic resonance imaging contrast agent. Biotinylated USPIO (bUSPIO) were targeted to Scavidin adenovirus-transduced HUVECs in vitro. Scavidin expressing cells were capable of binding and mediating endocytosis of the bUSPIO in vitro, which led to a significant decrease in T2 relaxation times, and a loss of signal intensity in comparison to controls. The findings were confirmed with Prussian blue staining for iron and detection of Scavidin by bound biotinylated horseradish peroxidase. Our data shows that biotinylated ligands target specifically to Scavidin-expressing HUVEC in vitro. The utilization of Scavidin gene transfer ex vivo thus constitutes a platform for potential ligand delivery via cell therapy and time-independent imaging of biologic processes.
