Discipline: Biological Sciences
LeKivia Cobb - University of Arkansas at Pine Bluff
Co-Author(s): Karl W. Boehme, University of Arkansas for Medical Sciences, Little Rock, AR
Mammalian orthoreovirus (reovirus) is a segmented double-stranded RNA virus that infects a wide range of hosts, including humans. Although most people become infected during early childhood, the majority infections are subclinical. However, reovirus is associated with disease in immunocompromised individuals and has recently been linked to the development of celiac disease. The goal of our work is to understand the viral determinants that underlie efficient reovirus replication. Previous work from our laboratory revealed that reovirus non-structural protein σ1s facilitates reovirus replication by promoting viral protein synthesis. However, the mechanism by which σ1s enhances reovirus protein synthesis is not known. One of the major roadblocks to understanding σ1s function is the lack of an antibody to detect σ1s. The goal of my project is to synthesize recombinant σ1s protein to use as an antigen for antibody production. Our approach is to use an inducible bacterial expression system to produce the σ1s protein. Past attempts to purify σ1s failed because the full-length protein was insoluble. Here, we divided the protein into two sections, an N-terminal fragment and a C-terminal fragment. We hypothesize that one half will be soluble and amenable to purification. The fragments were cloned into an IPTG-inducible system and 6x His or GST tags were added to facilitate purification. Under normal conditions σ1s is not synthesized, but after addition of IPTG σ1s will be produced. We will assess the induction and solubility of N- and C- terminal fragments of σ1s from two different reovirus strains, type 1 Long (T1L) and type 3 Dearing (T3D). For those constructs that are soluble, we will attempt to purify the protein using nickel- or GST- affinity chromatography. Once the protein is purified, it will be injected into rats to create an antibody that can be used for studies of σ1s. This reagent will help provide insight into how reoviruses translate their proteins in host cells.
Funder Acknowledgement(s): National Institutes of Health (NIH): 5R25HL108825-08 ; NIH: R01 AI118801 ; Center for Microbial Pathogenesis and Host Inflammatory Response COBRE: P20 GM103625
Faculty Advisor: Karl W. Boehme, KWboehme@uams.edu
Role: I conducted the part of research involving the transformation of constructs, IPTG induction, solubility testing, and and staining on SDS page gel.