Studies On Expression Of Human Zona Pellucida Protein 4 And Preparation Of Antibody And Binding To Sperm In Vitro

Objective: Zona pellucida(ZP),an extracellular matrix surrounding the mammalian oocyte,plays a key role in sperm-egg binding,inducing acrosome reaction and preventing spermiogenesis.The human zona pellucida consists of four zona pellucida proteins,ZP1,ZP2,ZP3 and ZP4,respectively.Among them,ZP4 plays an important role in sperm-egg specific identification and binding,preventing multiple inoculations,inducing acrosome reaction,ensuring ZP integrity,and protecting embryos and pre-implantation embryos.Human ZP4 is defined as the initial receptor that binds sperm,because of its important role in the initiation of sperm-egg binding and in the induction of exocytosis of sperm acrosome.ZP4 binds to the sperm cap and equatorial zone of the sperm-competent head,and ZP4 binding to the cap of the head is more than the ZP3.However,there have not been reported on the binding of hZP4 expressed in prokaryotic cells to non-capacitated sperm in vitro,and few domestic studies on the preparation of polyclonal antibodies against hZP4 is not published in China.Therefore,based on the biological characteristics both in ZP4 binding sperm cell membrane and their ligand interacting receptor,in this study,recombinant human ZP4 protein was used to bind with sperm in vitro and explore the binding efficiency of ZP4 to sperm,which provides a basis for the follow-up study of mixed spots in our group.The purified ZP4 protein was used to immunize New Zealand rabbits to obtain antiserum,which provided experimental basis for this study.Methods: The pGEX-4T-1-ZP4-His prokaryotic expression plasmid was constructed and induced to express GST-ZP4-His fusion protein in E.coli BL21(DE3),GST-ZP4-His fusion protein was purified by affinity chromatography with glutathione sepharose resin.Purified fusion protein was used to immunize New Zealand rabbits to prepare antiserum.The antiserum titer and specificity were determined by ELISA and Western blot.Purified ZP4 fusion protein binds to uncapacitated sperm in vitro,ZP4 co-localization with sperm were observed under fluorescence microscope.The binding efficiency of ZP4 to sperm was verified by inhibition experiment.Results: 1.The GST-ZP4-His fusion protein was successfully expressed in E.coliBL21(DE3).Specific GST-ZP4-His fusion protein was harvested after purification.2.A polyclonal anti-ZP4 antiserum was obtained after the fusion protein was used to immunize New Zealand rabbits.3.Immunofluorescence assay showed that prokaryotic recombinant human ZP4 protein did not bind to uncapacitated sperm in vitro.Inhibition experiment results showed that the curves before and after binding of ZP4 protein with uncapacitated sperm were similar.Conclusion: 1.The prokaryotic expression of GST-ZP4-His fusion protein was successfully obtained.2.The rabbit anti-human ZP4 antiserum had high titer but poor specificity.3.Recombinant human ZP4 protein produced from prokaryotic expression found no binding to uncapacitated sperm in vitro.