Date of Award
4-1-2021
Document Type
Master's Thesis - Open Access
Degree Name
Master of Science
Department
Microbiology and Immunology
First Advisor
Dana Mordue, Ph.D.
Abstract
Babesia microti is a tick born, intraerythrocytic parasite that is endemic to many North American regions. B. microti infection causes a disease called babesiosis, a disease whose clinical manifestation causes a wide range of symptoms, from asymptomatic infection to fulminant disease and possible death. Transfusion transmitted babesiosis is becoming an increasing concern as B. microti prevalence continues to grow and donor screening methods are ineffective. Transfusion transmitted babesiosis can be extremely dangerous, with as many as one in five cases resulting in death. Babesia microti secreted antigen (BmSA1) is an immunogen and reliable biomarker for B. microti infection. The aim of this work is to generate a recombinant BmSA1 protein using a trademarked Pichia pastoris expression system called “PichiaPink™”, for use in the development in an antigen specific ELISA as a protein standard. We hypothesize that expression of recombinant BmSA1 in a eukaryotic yeast system will better preserve the native confirmation and antibody recognition that might have been lost during expression in a prokaryotic expression system. We also use bioinformatics to assess the conservation of homology of the BmSA1 protein between all B. microti strain genomes published in piroplasmaDB.com. During this work PichiaPink™ yeast strains were successfully cloned with secretion vectors containing the bmsa1 gene. Probing culture supernatants by Western Blot analysis with hybridoma produced monoclonal antibodies was able to detect the rBmSA1 protein in culture supernatants, but the antibodies also recognize proteins other than rBmSA1 in the culture supernatant as well. Last, bioinformatics analysis of the BmSA1 protein, together with other species of the BMN family of proteins showed great conservation of the BmSA1 protein across ix all strains published in the piroplasmaDB.com database, suggesting the BmSA1 protein will be a reliable biomarker as the target of an antigen specific ELISA, as it does not appear to be subject to genetic variation across strain.
Recommended Citation
Centone, Anthony, "Heterologous expression of Babesia microti secreted antigen (BmSA1) in a Pichia pastoris expression system for the development of an antigen specific ELISA" (2021). NYMC Student Theses and Dissertations. 19.
https://touroscholar.touro.edu/nymc_students_theses/19
