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Polyomavirus EGFP-pseudocapsids: analysis of model particles for introduction of proteins and peptides into mammalian cells
Authors:Boura E  Liebl D  Spísek R  Fric J  Marek M  Stokrová J  Holán V  Forstová J
Affiliation:Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vinicná 5, 128 44 Prague 2, Czech Republic.
Abstract:
A vector for preparation of mouse polyomavirus capsid-like particles for transfer of foreign peptides or proteins into cells was constructed. Model pseudocapsids carrying EGFP fused with the C-terminal part of the VP3 minor protein (EGFP-VLPs) have been prepared and analysed for their ability to be internalised and processed by mouse cells and to activate mouse and human dendritic cells (DC) in vitro. EGFP-VLPs entered mouse epithelial cells, fibroblasts and human and mouse DC efficiently and were processed by both, lysosomes and proteasomes. Surprisingly, they did not induce upregulation of DC co-stimulation molecules or maturation markers in vitro; however, they did induce interleukin 12 secretion.
Keywords:AcNPV, Autographa californica nuclear polyhedrosis virus   BMDC, bone marrow-derived dendritic cells   CFSE, carboxyfluorescein diacetate succinimidyl ester   CM, complete culture medium   DC, immature human monocyte-derived dendritic cells   EEA1, early endosomal antigen   EGFP, enhanced green fluorescent protein   FCS, foetal calf serum   FSC and SSC, forward and side scatter characteristics   EM, electron microscopy   GM-CSF, granulocyte macrophage-colony stimulating factor   LAMP2, lysosomal associated membrane protein   LPS, lipopolysaccharide   NMuMG, normal murine mammary gland cells   p.a., post adsorption   p.i., post infection   PFU, plaque forming unit   poly(I:C), inosine-cytosine dsRNA polynucleotide   PyV, mouse polyomavirus   t-VP3, truncated, C-terminus of VP3 minor capsid protein   VLPs, empty artificial virus-like particles   VP1-VLPs, pseudocapsids composed of VP1 major capsid protein   EGFP-VLPs, pseudocapsids composed of VP1 major capsid protein and EGFP-t-VP3 fusion protein
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