Date of Award

Winter 2001

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

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.

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.

DOI

10.25777/xdtf-hg48

ISBN

9780493565170

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