Phage

Disponível somente no TrabalhosFeitos
  • Páginas : 36 (8771 palavras )
  • Download(s) : 0
  • Publicado : 8 de fevereiro de 2013
Ler documento completo
Amostra do texto
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1999, p. 3279–3286 0099-2240/99/$04.00 0 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 65, No. 8

Identification of Mimotope Peptides Which Bind to the Mycotoxin Deoxynivalenol-Specific Monoclonal Antibody
QIAOPING YUAN,1† JAMES J. PESTKA,2 BRANDON M. HESPENHEIDE,3 LESLIE A. KUHN,3 JOHN E. LINZ,2 AND L. PATRICKHART1* Departments of Botany and Plant Pathology,1 Food Science and Human Nutrition,3 and Biochemistry,2 Michigan State University, East Lansing, Michigan 48824
Received 23 October 1998/Accepted 6 May 1999

Monoclonal antibody 6F5 (mAb 6F5), which recognizes the mycotoxin deoxynivalenol (DON) (vomitoxin), was used to select for peptides that mimic the mycotoxin by employing a library of filamentousphages that have random 7-mer peptides on their surfaces. Two phage clones selected from the random peptide phage-displayed library coded for the amino acid sequences SWGPFPF and SWGPLPF. These clones were designated DONPEP.2 and DONPEP.12, respectively. The results of a competitive enzyme-linked immunosorbent assay (ELISA) suggested that the two phage displayed peptides bound to mAb 6F5specifically at the DON binding site. The amino acid sequence of DONPEP.2 plus a structurally flexible linker at the C terminus (SWGPFPFGGGSC) was synthesized and tested to determine its ability to bind to mAb 6F5. This synthetic peptide (designated peptide C430) and DON competed with each other for mAb 6F5 binding. When translationally fused with bacterial alkaline phosphatase, DONPEP.2 bound specifically tomAb 6F5, while the fusion protein retained alkaline phosphatase activity. The potential of using DONPEP.2 as an immunochemical reagent in a DON immunoassay was evaluated with a DON-spiked wheat extract. When peptide C430 was conjugated to bovine serum albumin, it elicited antibody specific to peptide C430 but not to DON in both mice and rabbits. In an in vitro translation system containing rabbitreticulocyte lysate, synthetic peptide C430 did not inhibit protein synthesis but did show antagonism toward DON-induced protein synthesis inhibition. These data suggest that the peptides selected in this study bind to mAb 6F5 and that peptide C430 binds to ribosomes at the same sites as DON. Deoxynivalenol (DON) (vomitoxin) (Fig. 1A) is one of the sesquiterpene mycotoxins classified as12,13-epoxy-trichothecenes (28). This compound occurs naturally in infected corn (19, 29), small grains (18, 31), and mixed feeds (29). DON is mainly produced by the fungus Gibberella zeae (Schwein.) Petch (anamorph, Fusarium graminearum Schwabe). At the cellular level, the main toxic effect of DON is inhibition of protein synthesis via binding to ribosomes and interfering with peptidyltransferase (4, 42). Inanimals, DON can cause anorexia and emesis (vomiting) (37). Other toxic effects of DON include skin irritation, hemorrhaging, hematological changes, human lymphocyte blastogenesis impairment, radiomimetic effects, apoptosis (cytotoxicity), and immunotoxicity (37). A major way to eliminate DON from human and animal food is to detect contaminated raw materials and divert them from feed and finishedfood. Compared with other analytic methods, immunoassays have several advantages for rapid field testing, including high specificity, sensitivity, facile sample preparation, and ease of use (33). Following the development of the first monoclonal antibody (mAb) to DON (6), immunological methods, mainly an enzyme-linked immunosorbent assay (ELISA), have been used widely for detection of DON (33).Unfortunately, the efficiency of chemical conjugation of DON to a carrier protein or an enzyme is low because such conjugation involves extensive modification and blocking stages and causes substantial bridge group interference and
* Corresponding author. Mailing address: Department of Botany and Plant Pathology, Michigan State University, East Lansing, MI 48824. Phone: (517) 353-9428. Fax: (517)...
tracking img