Journal: Frontiers in Microbiology
Article Title: Characterization of Plasmodium vivax Proteins in Plasma-Derived Exosomes From Malaria-Infected Liver-Chimeric Humanized Mice
doi: 10.3389/fmicb.2018.01271
Figure Lengend Snippet: Validation of PVX110940 in plasma-derived exosomes from P. vivax infected FRG huHep mice. (A) Schematic representation of full-length PVX110940 protein and a recombinant GST-fusion truncated (GST-PVX110940-Tr) version containing a predicted B-cell antigenic region of 156 amino acids. Red square represents a predicted N-terminal signal peptide. (B) GST-PV110940-Tr was produced from two E. coli clones (C11 and C25) in a wheat germ cell-free system and affinity-purified products resolved on SDS-PAGE. Western blot analysis using monoclonal anti-GST antibodies. (C) Recombinant GST-PVX110940-Tr was used to immunize mice and produce polyclonal antibodies. Western blot analysis shows recognition of GST-PVX110940-Tr purified recombinant protein and purified GST. (D) Flow cytometry bead-based assay showing PVX110940 detection in ExEF from plasma of P. vivax infected FRG huHep mice from EI1 and EI2. Beads were incubated with the highest-CD5L SEC fraction of each sample and used for detection of PVX110940 using the polyclonal antibodies raised in mice. Mean fluorescence intensity (MFI) of PVX110940 and controls was assayed. Specificity controls were as follows: C1: Highest-CD5L SEC fraction of each sample incubated with Alexa-488 mouse secondary antibody. C2: Pool of CD5L highest fraction of all samples incubated with isotype rabbit antibody and Alexa-488 mouse secondary antibody. Stars indicate the samples were positive detection was observed over the background signal observed in uninfected mice (Dashed line). (E) Western blot analysis of PVX-110940- in isolated exosomes (CD5L-highest SEC fraction) derived from plasma of P. vivax infected FRG huHep mice from EI1 and EI2. Hundred micro liters of CD5L-highest fraction were blotted. Membranes were incubated with 1/50 of polyclonal anti-PVX110940 antibody. * Samples where a positive signal at the expected molecular size (84 kDa) is observed.
Article Snippet: GST-PVX110940-Tr recombinant protein was purified by affinity chromatography on GST spin trap columns (GE healthcare) and eluted with 10 mM glutathione in 50 mM Tris-HCl pH 8.0.
Techniques: Derivative Assay, Infection, Recombinant, Produced, Clone Assay, Affinity Purification, SDS Page, Western Blot, Purification, Flow Cytometry, Bead-based Assay, Incubation, Fluorescence, Isolation
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