DOI
https://doi.org/10.25772/362W-R260
Defense Date
1994
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Biochemistry
First Advisor
Darrell L. Peterson
Abstract
The hepatitis c virus (HCV) core gene has been chemically synthesized and used to direct the expression of core protein in Escherichia coli (E.coli). When cloned downstream of the inducible T7 promotor the core gene directed the expression of a soluble protein with a molecular weight of 22 kDa. Under native conditions the protein behaved in a manner consistent with the formation of a multimeric structure, which may represent assembled core particles. Further, when examined by electron microscopy a heterogeneous mixture of nucleocapsid-like particles were visible. Core protein was specifically recognized by antibodies present in HCV infected serum, suggesting that this protein may be useful as a diagnostic tool for detecting HCV infection. To circumvent the difficulty in purifying this protein in significant quantities a core-polyhistidine fusion gene was constructed. This protein was chromatography, readily purified, using nickel under denaturing conditions. chelation. Studies are currently on-going to ascertain the conditions required for refolding this protein, and the suitability of this protein to serve as a diagnostic tool.
In a second study, the major core protein (p26) of the lentivirus equine infectious anaemia (EIAV) was expressed in E.coli and purified to >95% homogeneity. Circular dichroism spectroscopy revealed that p26 exhibits the following assignment of secondary structural elements; 40% alpha helix, 22% beta sheet, 10% beta turn, and 28% random coil. It has been determined that p26 contains a single free cysteine residue (Cys48), and an intramolecular disulfide bond between cysteine residues 198 and 213. Data acquired by circular dichroism spectroscopy and fluorescence spectroscopy indicate that this disulfide bond plays a critical role in maintaining the structure of p26. Crystals of p26 were successfully grown at a protein concentration of 8mg/ml in 0.1M cacodylate, 0.7M sodium acetate, pH 6.5, at 4 °C. A sandwich ELISA was developed, using recombinant p26, to detect anti-p26 antibodies in horse sera. The assay successfully identified the 19 positive sera in a blind panel of 30 samples supplied by the National Veterinary Service Labs (Ames, Iowa). Accordingly, the assay appears to meet the criteria required of a commercial diagnostic reagent for determining EIAV infection in horses.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
7-14-2016