Thesis
Stable expression of GFP-BSD and surface exposed BM86 epitopes in transfected B. bovis merozoites
Washington State University
Master of Science (MS), Washington State University
2011
Handle:
https://hdl.handle.net/2376/101803
Abstract
Babesia bovis is a tick-born intraerythocytic protozoan causing acute hemolytic disease in cattle. Currently, vaccines based on live attenuated B. bovis parasites are the most effective preventative strategy against this disease because they invoke protective immune stimulation throughout the persistent infection of the host. Additionally, B-cell epitopes derived from Rhipicephalus (Boophilus) microplus tick midgut antigen Bm86 are able to elicit partial protection against tick challenges in vaccinated cattle. A live vaccine able to confer protection against both Babesia and its tick vector would be a practical and effective tool to improve control of these parasites. Recently, a stable transfection method for B. bovis has been developed, but the construct used for transfection is limited to a single expression site and the protein expressed induced negligible antibody responses in vaccinated cattle. To address the limitation of the single expression site, the ef-1a IG region was tested and confirmed to regulate two independent expression sites in transiently transfected B. bovis parasites using a luciferase assay. This bidirectional promoter was then used to develop a novel stable transfection vector for the expression of two independent exogenous genes: one encoding a chimera of the highly antigenic surface exposed protein MSA-1 with Bm86 B-cell epitopes, and the other with the reporter and selection fusion gene gfp-bsd. The plasmid was transfected into the biologically cloned and attenuated Mo7 strain of B. bovis and shown to integrate into the ef-1a locus by PCR and Southern hybridizations. Simultaneous expression of the two antigens and surface expression of Bm86 B-cell epitopes was demonstrated using western hybridization and immunofluorescence analyses. With the limitations of the previous transfection system resolved, it is now possible to use this novel transfection approach as a platform to test attenuated B. bovis as a vaccine delivery system in cattle.
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Details
- Title
- Stable expression of GFP-BSD and surface exposed BM86 epitopes in transfected B. bovis merozoites
- Creators
- Jacob M. Laughery
- Contributors
- Donald P. Knowles (Degree Supervisor)Carlos E. Suarez (Degree Supervisor)
- Awarding Institution
- Washington State University
- Academic Unit
- Veterinary Microbiology and Pathology, Department of
- Theses and Dissertations
- Master of Science (MS), Washington State University
- Publisher
- Washington State University; Pullman, Wash. :
- Identifiers
- 99900525037801842
- Language
- English
- Resource Type
- Thesis