Wai-Hong Wu

Document Type

Dissertation - University Access Only

Award Date

2002

Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology and Microbiology

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is an important infectious disease of swine and is responsible for significant economic losses worldwide. In order to develop more effective management and control strategies, our long term goal is to further characterize the viral proteins and define their functions. Therefore the objectives stated in this thesis include: 1) expression of different genes of PRRSV, 2) generation of PRRSV protein specific monoclonal antibodies (MAbs), and 3) identification and 4) characterization of the PRRSV structural proteins. During this study, panels of recombinant proteins and MAbs were generated to serve as tools for the further characterization of the PRRSV proteins. A new, 10 kDa structural protein of PRRSV, designated 2b, was identified. This protein is expressed as the principle product of mRNA2, even though its translation initiation codon is not in the first position on the mRNA. Using a MAb developed against the PRRSV 2b protein, another protein with an approximate size of 14 kDa was co-immunoprecipitated with 2b and may represent the PRRSV nucleocapsid protein. A different MAb against the nucleocapsid protein co-immunoprecipitated a protein with a approximate size of 1 O kDa. Therefore, it is possible that the 2b and nucleocapsid proteins may interact and play a key role in viral morphogenesis. For further characterization of the PRRSV structural proteins, a baculovirus / mammalian cell expression system was established. Individual PRRSV proteins were expressed in MARC-145 cells transduced with recombinant baculoviruses, and the expressed proteins demonstrated subcellular localization patterns similar to those previously reported in the literature. When the 2b protein was expressed in the baculovirus / mammalian expression system with a fluorescent tag, the formation of inclusion bodies was frequently observed. However, the signal in a zeocin-selected MARC-145 cell line had a low intensity during normal cell division and growth, while a high intensity signal was apparent during cellular deterioration. Further investigation is needed to understand whether expression of the 2b protein is related to the impairment of cellular protein degradation pathways, such as the ubiquitin-proteasome system. Finally, a baculovims / mammalian cell expression system-based microarray assay was developed and a set of potential differentially expressed genes was identified.

Library of Congress Subject Headings

Swine -- Virus diseases Viral proteins Porcine reproductive and respiratory syndrome

Format

application/pdf

Number of Pages

253

Publisher

South Dakota State University

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