Document Type

Thesis - Open Access

Award Date

2015

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Xiuqing Wang

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped, single stranded, positive sense RNA virus and a member of Arteriviridae. Its genome encodes 10 open reading frames for at least 7 structural proteins and 14 non-structural proteins. Membrane (M), Nuclepcapsid (N), and Glycoprotein-5 (GP5) are the major structural proteins of PRRSV, while Envelope (E), Glycoprotein-2 (GP2), Glycoprotein-3 (GP3), and Glycoprotein-4 (GP4) are the minor structural proteins of PRRSV. GP5 induces neutralizing antibodies and forms heterodimers with M, while N is the most immunogenic protein of PRRSV. Previous studies have shown viral structural proteins are able to form virus-like particles (VLPs) that can induce an immune response in respective hosts by use of the recombinant baculovirus expression system in insect cells. We sought to find if structural proteins, M, N, GP5, GP4, and E, could be expressed using the recombinant baculovirus expression system and produce VLPs. After purifying and amplifying PRRSV GP4, GP5, and E genes from PRRSV viral RNA, we inserted those genes into the pCAGEN plasmid, and expressed them in mammalian cells (cos-1). Protein expression of E and GP5 in both the supernatant and cell lysate was verified through western blot, while GP4 was not. We inserted the expressed genes into the pOET-1 plasmid for production of recombinant baculoviruses x using the flashBAC expression system in insect cells. Expression of GP5 and E proteins were verified with IFA. Viral titers were collected using bacuQUANT qRT-PCR, and used to co-infect TriEx SF9 cells with recombinant baculoviruses containing PRRSV M, N, GP5, and E at a multiplicity of infection (MOI) of 2 for N, GP5, and E proteins and a MOI of 3 for M protein for 72 hours. Protein expression was confirmed through western blot. The results of this experiment show that PRRSV M, N, GP5, and E proteins are expressed after co-infection of TriEx SF9 cells using the recombinant baculovirus system, and that VLPs of similar shape and size to the PRRSV virion are produced. These results will aid in further research of vaccine production using PRRSV VLPs

Library of Congress Subject Headings

Porcine reproductive and respiratory syndrome
Swine--Virus diseases
Baculoviruses
Recombinant proteins
Viral vaccines

Description

Includes bibliographical references (pages 56-65)

Format

application/pdf

Number of Pages

75

Publisher

South Dakota State University

Download

Share

COinS
 

Rights Statement

In Copyright