Date of Award:

5-1-1998

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biology

Committee Chair(s)

Joseph K.-K. Li

Committee

Joseph K.-K. Li

Committee

Jon Y. Takemoto

Committee

Gregory J. Podgorski

Abstract

The NS2 protein of bluetongue virus (BTV) serotype 17 (B17) was expressed in large scale as a Glutathione-S-Transferase (GST) fusion protein in E. coli cells. The size of the expressed NS2/GST fusion protein was about 68 kilodaltons (kDa) when analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). This is equal to the sum of the predicted size from the deduced amino acid sequence of the NS2 protein (41 kDa) and the known mass of the GST protein (27 kDa). The NS2/GST fusion protein was purified by absorption to glutathione-conjugated agarose beads (G-beads), and then analyzed on western blots by two monospecific oligoclonal antibodies (OAbs). Two linear antigenic domains were mapped by the same two OAbs, which were elicited by two synthetic oligopeptides. The NS2 protein was also expressed in Sf-9 cells using the baculovirus expression system. The expressed protein had a molecular mass of 41 kDa, which is consistent with the predicted size from the deduced amino acid sequence. The identity of the expressed NS2 protein was also confirmed by the two OAbs. NS2 expressed in E. coli and Sf-9 cells possessed single-stranded RNA (ssRNA) binding activity towards ssRNA transcripts from bluetongue viral genes and to ssRNAs extracted from yeast, as well as to poly(U) conjugated to Sepharose 4B resin. The binding of the NS2/GST fusion protein to each RNA species was concentration dependent but not sequence specific. Three synthetic peptides, derived from the deduced amino acid sequence of NS2 and located between #2-11, #153-166, and #274-286, exhibited ssRNA binding activity. Two of these peptides also represent two of the antigenic determinants of the NS2 protein, and they are located at the C-terminal part of the NS2 protein. Several truncated NS2 proteins were expressed and partially purified. The ssRNA binding abilities of these truncated proteins were different from that of the intact NS2/GST fusion protein. Electrophoresis mobility shift assays (EMSA) indicated that two domains within the N-terminal half and one domain at the C-terminal end of the NS2 protein were primarily responsible for its ssRNA binding activity.

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