Date of Completion

Spring 5-8-2011

Thesis Advisor(s)

Guillermo Risatti

Department

Animal Science

Disciplines

Animal Sciences | Other Animal Sciences

Abstract

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is single-stranded, positive-sense RNA virus in the family Arteviridae, order Nidovirales. PRRSV began circulating in the U.S swine population in the 1980's and since then becoming the most economically significant disease of swine herds. PRRSV is now disseminated worldwide causing great economic losses to pork industries. PRRSV single-stranded RNA genome is15 kb in length. The genome encodes 9 open reading frames (ORF1a, ORF1b,

ORF2a, ORF2b and ORFs 3 through 7). ORFs 1a and 1b encode for 13 non-structural proteins (nsp) that are suggested to be involved in transcription and viral genome replication. The exact role of non-structural proteins in PRRSV cycle is still unknown. Moreover, there is a limited availability of reagents, such as antibodies against these non-structural proteins, that further limits their study. To overcome that limitation, the gene encoding for non-structural protein 12 (nsp-12) was synthesized using the polymerase chain reaction (PCR) and cloned in-frame into a plasmid vector (pRSET A) to create the plasmid construct pRSETAnsp-12. After transforming Escherichia coli with pRSETAnsp-12, individual colonies that grew in the presence of ampicillin were selected, cultured in terrific broth-ampicillin media, and plasmid DNA extracted to evaluate for the presence and fidelity of the nsp-12 gene by means of agarose gel electrophoresis separation and sequencing. DNA plasmids harboring a correct copy of the gene were used to express nsp-12 protein in BL21 competent Escherichia coli cells. After growth of BL21 transformants in "Magic media"(r), expression of nsp-12 was assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) separation followed by Coomassie blue staining and western blot detection using an anti-His monoclonal antibody. PRRSV nsp-12 was then purified by metal affinity chromatography. Purified nsp-12 protein will be used to produce an anti-nsp-12 polyclonal antibody serum in rabbits for further study the functions of this protein.