Anzucht, Aufreinigung und partielle Charakterisierung von Kleinen Virus-ähnlichen Partikeln des JC-Virus

Abstract

Virus-like particles are viral capsids without virus-specific nucleid acids. They are produced by recombinant expression of the structural proteins of the virus and are free of viral nucleid acids. Therefore, they are not virus-typically pathogenic and thus a promising model for vaccine development and gene therapeutics. The virus-like particles used in this thesis consist of the major structural protein VP1 of JC-Virus, a member of the Polyomavirus family. The capsid of polyomaviruses normally consists of the structural proteins VP1, VP2 and VP3, in which VP1 dominates the assembly and forms the basic structure which consists of 72 VP1-pentamers. Therefore, recombinant expressed VP1 is capable to self-assemble into virus-like particles. Beside normal virus-like particles, sized like the wild-type virus with a diameter of 45 nm, small virus-like particles with a diameter of 20 - 25 nm are also generated and are the main topic of this thesis. These were enriched by caesium chloride gradient centrifugation within the fraction of 1,31 g/mL density. In addition to electron microscopy as the usual detection method, a native gel electrophoresis, which is able to differentiate between small and normal virus-like particles, was developed. Furthermore, small virus-like particles were dissociated under low calcium reducing conditions into their basic structural elements; so and called VP1- pentamers. It was shown that these have an analogous reactivity to normal-sized virus-like particles. Moreover, it was shown that, with the addition of nucleid acids, small virus-like particles converted into normal-sized particles through dissociation and reassociation processes. This pattern is of particular interest for the industrial production of virus-like particles. Therefore, small virus-like particles do not necessarily represent a contamination of normal-sized virus like particles and can be purified through the same processes. Due to their small size, additional specific applications such as transport proteins for active pharmaceutical agents might be possible.