VTechWorks staff will be away for the Thanksgiving holiday beginning at noon on Wednesday, November 27, through Friday, November 29. We will resume normal operations on Monday, December 2. Thank you for your patience.
 

The localization of two epitopes recognized by the monoclonal antibody PCG-4 on toxin A of Clostridium difficile

Simple item page
dc.contributor.authorFrey, Steven M.en
dc.contributor.committeechairWilkins, Tracy D.en
dc.contributor.committeememberJohnson, John L.en
dc.contributor.committeememberChen, Jiann-Shinen
dc.contributor.departmentMicrobiologyen
dc.date.accessioned2014-03-14T21:35:20Zen
dc.date.adate2009-05-02en
dc.date.available2014-03-14T21:35:20Zen
dc.date.issued1990-12-05en
dc.date.rdate2009-05-02en
dc.date.sdate2009-05-02en
dc.description.abstractClostridium difficile causes pseudomenbranous colitis (PMC) and diarrhea in humans. Toxigenic strains of C. difficile produce two toxins. Toxin A is an enterotoxin and cytotoxin, and toxin B is a potent cytotoxin. The gene encoding toxin A has been sequenced and was shown to possess a 2.5 kb region, containing 38 similar repeating amino acid sequences, at the 3' -end of the gene. This region of the toxin A gene codes for the carbohydrate-binding portion of the toxin. The monoclonal antibody PCG-4 (MAb) binds to this portion of toxin A and neutralizes its enterotoxic activity. In addition, this monoclonal antibody has been shown to immunoprecipitate toxin A, suggesting that the MAb PCG-4 is binding to two or more similar epitopes on the toxin. The goal of this research project was to identify the neutralizing epitopes recognized by the MAb PCG-4 on the surface of the toxin A. To map the epitopes bound by the MAb PCG-4, a series of overlapping deletion clones were constructed from a 4.7 kb fragment from the 3'-end of the toxin A gene. The recombinant polypeptides expressed by these clones were tested for reactivity with the MAb PCG-4. By comparing the overlapping polypeptides, defined as either PCG-4 reactive or nonreactive, I localized the PCG-4 epitope to a 44-amino acid sequence situated between the amino acid residues 2098-2141 of toxin A A similarity search of the toxin with the 44-amino acid sequence containing the PCG-4 epitope revealed the presence of two other possible PCG-4 epitopes located between the amino acid residues 2355-2398 and 2459-2502. However, subsequent cloning experiments showed that only the region located between the amino acid residues 2355-2398 contained a PCG-4 reactive epitope. The identification of two similar epitopes within the toxin's structure explains how this monoclonal antibody is able to immunoprecipitate toxin A in the absence of subunits. Furthermore, I found that small recombinant polypeptides, containing the PCG-4 epitope lost reactivity with this monoclonal antibody following denaturation, suggesting that the epitopes recognized by this monoclonal antibody are conformationally dependent.en
dc.description.degreeMaster of Scienceen
dc.format.extentvi, 99 leavesen
dc.format.mediumBTDen
dc.format.mimetypeapplication/pdfen
dc.identifier.otheretd-05022009-040613en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-05022009-040613/en
dc.identifier.urihttp://hdl.handle.net/10919/42409en
dc.language.isoenen
dc.publisherVirginia Techen
dc.relation.haspartLD5655.V855_1990.F749.pdfen
dc.relation.isformatofOCLC# 23812421en
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subject.lccLD5655.V855 1990.F749en
dc.subject.lcshClostridium difficile -- Researchen
dc.titleThe localization of two epitopes recognized by the monoclonal antibody PCG-4 on toxin A of Clostridium difficileen
dc.typeThesisen
dc.type.dcmitypeTexten
thesis.degree.disciplineMicrobiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

Files

Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
LD5655.V855_1990.F749.pdf
Size:
11.94 MB
Format:
Adobe Portable Document Format
Download

Collections

Masters Theses