The Experimental Study On The Expression Of Recombinant Human Zona Pellucida Protein 3, Preparation Of Antibody And Binding With Sperm In Vitro

Objective:The recognition of mammalian gametes is initiated from the binding of sperm to the zona pellucida(ZP).The human ZP consists of four glycoproteins,named ZP1,ZP2,ZP3,and ZP4.Among them,human zona pellucida glycoprotein 3(h ZP3),as the initial receptor for sperm-egg binding,also showed strong binding ability to spermatozoa while inducing sperm acrosome reaction.Due to the difficulty in obtaining natural h ZP3,most studies have focused on the binding of recombinant h ZP3 to capacitated spermatozoa in vitro.There is no relevant research report on the binding of prokaryotic expression h ZP3 to unstimulated sperm in vitro.And at the same time,there has not been published on producing anti-h ZP3 for the whole amino acid in China.Therefore,based on the characteristics both in sperm-egg binding and the interaction of receptor-ligands,this study intends to produce recombinant h ZP3 in prokaryotic expression system and subsequently perform on combination with unstimulated spermatozoa.Based on above method,it will be applied to isolate sperms from the mixed stains in the forensic science field in the following study.In addition,the purified recombinant h ZP3 will be used as an antigen to generate anti-h ZP3 antiserum,which will provide the material basis for subsequent experiments.Methods:The h ZP3 gene was cloned into the prokaryotic expression vector p GEX-4T-1,and the h ZP3-GST-His fusion protein was expressed by inducing E.coli;the protein was purified by affinity chromatography on glutathione agarose resin.New Zealand white rabbits were immunized with purified h ZP3 to prepare antiserum.The titer and specificity of rabbit antiserum were examined by ELISA and Western blot analyses.A binding assay between the purified h ZP3 and unstimulated sperm was carried out.The co-localization of h ZP3 and sperm was observed by immunofluorescence technique.The binding efficiency of h ZP3 to sperm was examined by inhibition experiment.Results:1.The hZP3-GST-His fusion protein was expressed in E.coli successfully;a specific recombinant h ZP3 with a molecular weight of approximately 73 k Da was obtained;2.The purified h ZP3 was used to immunize New Zealand white rabbits and a high titer of anti-h ZP3 protein was obtained.But the polyclonal antiserum was poor in specificity;3.Immunofluorescence results showed that the recombinant h ZP3 expressed in prokaryotic cells was rare bound to unstimulated spermatozoa in vitro,but inhibition experiments showed that the OD curve after h ZP3 protein reacting with sperms had a decreasing trend compared with one before binding.Conclusion:Recombinant h ZP3 was successfully expressed by the E.coli expression system and purified;rabbit anti-rh ZP3 prepared by immunize rabbits had high titer,but the specificity was poor;recombinant h ZP3 showed weaker binding to un-capacitated human spermatozoa in vitro.