Date of Award
Winter 2001
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Program/Concentration
Biomedical Sciences
Committee Director
Ke-Wen Dong
Committee Member
Sergio Oehninger
Committee Member
Frank Castora
Committee Member
Michael Solhaug
Abstract
Despite numerous reports indicating the successful production of bioactive recombinant ZP3, no report has shown the rhZP3 having direct binding activity with human sperm. Recombinant ZP3 generated from our previous study displayed binding activity with human sperm through indirect evidence from hemizona assay (HZA).
This present study focused on the production of recombinant ZP3 with direct binding activity with human sperm. Through the application of a pEGFP expression vector, fusion protein GFP/ZP3 was successfully generated and expressed. The expression of GFP/ZP3 was evidenced by RT-PCR and western blot. The fusion protein was partially purified by Ni-NTA affinity column from cell culture medium of stably transfected cells generated from a single cell clone. Immuno-blotting analysis of the fusion protein indicated that the GFP/ZP3 was about 90–92 kD in molecular mass which is close to the sum of the size of GFP and ZP3. Flow cytometry was conducted to evaluate the direct binding activity of fusion protein with human sperm. The GFP/ZP3 showed dose-dependence in the binding assay. GFP tag removal resulted in almost no detectable binding as assayed by flow cytometry, thereby indicating that ZP3 binding was specific. Furthermore, immunofluorescence demonstrated that the fusion protein interacted with human sperm on the acrosome region. In the acrosome reaction assay, fusion protein was proved to be able to induce the sperm acrosome reaction (19.2% ± 3.4% as compared to basal line 10.8% ± 2.3%) at the concentration of 1ug/ml. Therefore, the fusion protein displayed full spectrum biological activity of ZP3, and, most importantly unlike prior studies, this binding activity was elucidated in a direct way.
DOI
10.25777/xdtf-hg48
ISBN
9780493565170
Recommended Citation
Lin, Zhiyong.
"Characterization of the Biological Activities of Recombinant Fusion Protein Green Fluorescent Protein/Human Zona Pellucida Protein 3 (GFP/HZP3)"
(2001). Doctor of Philosophy (PhD), Dissertation, , Old Dominion University, DOI: 10.25777/xdtf-hg48
https://digitalcommons.odu.edu/biomedicalsciences_etds/54
Included in
Biomedical Commons, Cell Biology Commons, Molecular Biology Commons
Comments
Dissertation submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.