Date of Completion

Spring 5-8-2011

Thesis Advisor(s)

James Cole

Honors Major

Cell Biology

Disciplines

Cell Biology | Molecular Biology

Abstract

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is single-stranded, positive-sense RNA virus in the family Arteriviridae, order Nidovirales. PRRSV is the most economically significant viral infection of swine herds in the United States. The single-stranded RNA genome is 15 kb in length and 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 coding for non-structural protein 3 (nsp-3) was synthesized using the polymerase chain reaction (PCR) and cloned in-frame into a plasmid vector to create a construct named pRSETA-nsp3. After transforming Escherichia coli with pRSETA-nsp3, individual colonies that grew in the presence of ampicillin were selected. These colonies were cultured in terrific broth-ampicillin media. Then, plasmid DNA was extracted to evaluate for the presence and fidelity of the nsp-3 gene by means of agarose gel electrophoresis separation and sequencing. DNA plasmids harboring a correct copy of the gene were used to express nsp-3 protein in BL21 competent Escherichia coli cells. After growth of BL21 transformants in "Magic media"(r), expression of nsp-3 was assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) separation followed by Coomassie blue staining and western blot detection using an anti His-tag monoclonal antibody. PRRSV nsp-3 was then purified by metal affinity chromatography. Purified nsp-3 protein will be used to produce an anti nsp-3 polyclonal antibody serum in rabbits for further study of the functions of this protein.

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