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      • Development of a novel peptide microarray for large-scale epitope mapping of food allergens

        Lin, Jing,Bardina, Ludmilla,Shreffler, Wayne G.,Andreae, Doerthe A.,Ge, Yongchao,Wang, Julie,Bruni, Francesca M.,Fu, Zhiyan,Han, Youngshin,Sampson, Hugh A. Elsevier 2009 The Journal of allergy and clinical immunology Vol.124 No.2

        <P><B>Background</B></P><P>The peptide microarray is a novel assay that facilitates high-throughput screening of peptides with a small quantity of sample.</P><P><B>Objective</B></P><P>We sought to use overlapping peptides of milk allergenic proteins as a model system to establish a reliable and sensitive peptide microarray-based immunoassay for large-scale epitope mapping of food allergens.</P><P><B>Methods</B></P><P>A milk peptide microarray was developed by using commercially synthesized peptides (20-mers, 3 offset) covering the primary sequences of α<SUB>s1</SUB>-casein, α<SUB>s2</SUB>-casein, β-casein, κ-casein, and β-lactoglobulin. Conditions for printing and immunolabeling were optimized using a serum pool of 5 patients with milk allergy. Reproducibility of the milk peptide microarray was evaluated using replicate arrays immunolabeled with the serum pool, whereas specificity and sensitivity were assessed by using serial dilution of the serum pool and a peptide inhibition assay.</P><P><B>Results</B></P><P>Our results show that epitopes identified by the peptide microarray were mostly consistent with those identified previously by SPOT membrane technology, but with specific binding to a few newly identified epitopes of milk allergens. Data from replicate arrays were reproducible (<I>r</I> ≥ 0.92) regardless of printing lots, immunolabeling, and serum pool batches. Using the serially diluted serum pool, we confirmed that IgE antibody binding detected in the array was specific. Peptide inhibition of IgE binding to the same peptide and overlapping peptides further confirmed the specificity of the array.</P><P><B>Conclusion</B></P><P>A reliable peptide microarray was established for large-scale IgE epitope mapping of milk allergens, and this robust technology could be applied for epitope mapping of other food allergens.</P>

      • A Gene-Shuffled Glyphosate Acetyltransferase Protein from Bacillus licheniformis (GAT4601) Shows No Evidence of Allergenicity or Toxicity

        Delaney, Bryan,Zhang, John,Carlson, Gabrielle,Schmidt, Jean,Stagg, Barb,Comstock, Brad,Babb, Amy,Finlay, Carol,Cressman, Robert F.,Ladics, Greg,Cogburn, Amarin,Siehl, Dan,Bardina, Luda,Sampson, Hugh,H Oxford University Press 2008 Toxicological sciences Vol.102 No.2

        <P>The glyphosate acetyltransferase (gat) gene from Bacillus licheniformis was subjected to multiple rounds of gene shuffling to optimize kinetics of corresponding GAT proteins to acetylate the herbicide active ingredient glyphosate. Genetically modified soybeans expressing the gat4601 gene (356043 soybeans) are tolerant to the application of glyphosate. The current manuscript reports the outcome of the allergenicity and toxicity assessment for the GAT4601 protein. Bioinformatic comparison of the amino acid sequence of GAT4601 did not identify similarities to known allergenic or toxic proteins. In vitro studies conducted with heterologously produced GAT4601 protein demonstrated that it was rapidly degraded in simulated gastric fluid containing pepsin (< 30 s) and in simulated intestinal fluid containing pancreatin (< 2 min) and completely inactivated at temperatures above 56 degrees C. The GAT4601 protein expressed in planta is not glycosylated and similar protein profiles were observed in flour extracts from 356043 soybeans and nontransgenic near isoline comparator soybeans (Jack) using serum from soy allergic persons. No evidence of adverse effects was observed in mice following acute oral exposure to 2000 mg/kg of GAT4601 protein or in a repeated dose dietary exposure study at doses of 800-1000 mg/kg/day. This comprehensive assessment demonstrates that the GAT4601 protein does not present a risk for adverse effects in humans when used in the context of agricultural biotechnology.</P>

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