Expression of recombinant porcine circovirus 2 (PCV2) capsid polypeptides for mapping antibody epitopes following vaccination, infection, and disease
| Autor: | Trible, Benjamin R. |
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| Rok vydání: | 2012 |
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| Druh dokumentu: | Diplomová práce |
| Popis: | Master of Science Department of Diagnostic Medicine/Pathobiology Raymond R. R. Rowland Open reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) codes for the 233 amino acid capsid protein (CP). Baculovirus-based vaccines that express only ORF2 are protective against clinical disease following experimental challenge or natural infection. The goal of this study was to identify regions in CP preferentially recognized by sera from experimentally infected and vaccinated pigs, and compare these responses to pigs diagnosed with porcine circovirus-associated disease (PCVAD). The approach was to react porcine sera with different CP polypeptide fragments that each contained one or more immunoreactive regions. Expression of polypeptides was performed using E.coli. Initial results showed that sera from vaccinated pigs preferentially recognized only the largest CP(43-233) polypeptide fragment and showed low levels of binding to other CP polypeptide fragments. The results of sera from pigs diagnosed with PMWS showed only minimal reactivity with CP polypeptide fragments, including the largest CP(43-233). PCV2 infected or PDNS diagnosed pigs reacted to all CP polypeptides: however, the strongest reactivity was primarily directed towards CP polypeptides containing residues in the 160-180 region. For this purpose, finer mapping studies were performed. These experiments involved reacting sera from experimentally infected PCV2 pigs and PDNS pigs with overlapping oligopeptides that covered amino acids 141-200. Overall, the results showed a subset of experimentally infected pigs and pigs with PDNS preferentially recognized the CP oligopeptide, 169-STIDYFQPNNKR-180. Alanine scanning identified Y-173, F-174, Q-175 and K-179 as important for antibody recognition. The results from this study support the notion of PCV2 modulation of immunity, including antibody responses that may represent a precursor for disease. The results from this study support the notion of PCV2 modulation of immunity. Furthermore, the methods incorporated in this study provide a means for characterizing the immune response upon vaccination, natural infection and disease. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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