Immunofluorescence analysis of baculovirus-displayed viral proteins on infected insect cells.
The baculovirus expression vector system has been used extensively to produce numerous proteins originating from both prokaryotic and eukaryotic sources. In addition to easy cloning techniques and abundant viral propagation, the system's insect cell environment provides eukaryotic post-translational modification machinery. The baculovirus display vector system provides a number of advantages over prokaryotic systems, allowing the combination of genotype with phenotype, enabling presentation of foreign peptides or even complex proteins on the baculoviral envelope or capsid. Baculoviruses permit larger gene insertions, are easily propagated, and can be grown to high titers. Furthermore, the eukaryotic system allows for post-translational modifications, and surface modifications of the viral capsid enable specific targeting. This strategy can be used to enhance viral binding and entry to a wide variety of both dividing and nondividing mammalian cells, as well as to produce antibodies against the displayed antigen. In addition, the technology should enable modifications of intracellular behavior, i.e., trafficking of recombinant "nanoparticles," a highly relevant feature for studies of targeted gene or protein delivery. After generating the display viral stock, it is important to confirm the presence and functionality of the displayed peptides or proteins on the viral particles before proceeding to further experiments. Accordingly, infected insect cells and budded virions can be analyzed by a variety of methods using appropriate antibodies. This protocol describes a standard immunofluorescence technique in detail.
Mäkelä
Anna R
AR
Ernst
Wolfgang
W
Grabherr
Reingard
R
Oker-Blom
Christian
C
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D005455:Fluorescent Antibody Technique / Q000379:methods; D015870:Gene Expression; D005822:Genetic Vectors; D007313:Insecta; D011994:Recombinant Proteins / Q000032:analysis*; D014764:Viral Proteins / Q000032:analysis*; D014771:Virion / Q000737:chemistry*
D011994:Recombinant Proteins; D014764:Viral Proteins