The Experimental Study On The Recombinant Human Zona Pellucida Protein 2 Binds To Sperm In Vitro As Well As Investigation Of 4 SNPs Loci Gene Polymorphism

Objective:Mammalian egg membrane glycoprotein?zona pellucida,ZP?is a kind of glycoprotein matrix secreted by oocytes and granulosa cells.Human zp is divided into ZP1,ZP2,ZP3 and ZP4,in which ZP2 is a secondary receptor involved in sperm and egg recognition.At present,most of the studies in reproductive field focus on the binding of ZP2 protein to capacitated spermatozoa,but few studies on binding to uncapacitated sperm,and the binding environment is more single than that of non-capacitated spermatozoa.In this study,the binding of recombinant hZP2 protein expressed in eukaryotic system to uncapacitated spermatozoa in different binding environments in vitro was investigated by using the affinity of receptor and ligand in vitro.It is expected that the recombinant protein can combine with sperm efficiently and be applied in forensic science,which will provide the basis for the extraction and personal recognition of sperm from mixed spot of forensic medicine in our research group.To explore potential genetic risk factors for Parkinson disease?PD?in the northern Han Chinese population and to provide a new genetic marker for forensic personal identification,3 single nucleotide polymorphisms?SNPs??rs7479949,rs2053288 and rs12612615?in DLG2,RBMS3 and NAP5 genes and 1 insertion/deletion marker?InDel??rs778533301?in NAP5 genes were investigated.Methods:The recombinant hZP2 protein was expressed in HEK293 cells by using the constructed pCDNA3.1-ZP2 eukaryotic recombinant plasmid and purified by Ni-NTA agarose affinity chromatography.By changing the number of spermatozoa,the concentration of Ca²⁺and the incubation time,the binding experiment of purified hZP2 and uncapacitated sperm was carried out in vitro.The co-localization of hZP2and sperm was detected by immunofluorescence technique.Inhibition experiments were used to detect the binding efficiency of hZP2 to sperm.Using polymerase chain reaction?PCR?and restriction fragment lengthpolymorphism?PCR-RFLP?,a total of 332 healthy individuals and 331 PD patients from northern China were enrolled in this study.The genetic polymorphisms of three snps loci and one indel locus were investigated,genetic and forensic parameters were calculated,and the data of control and case group were statistically analyzed.Results:1.The recombinant hZP2 protein was successfully expressed in HEK293cells and purified to obtain the recombinant hZP2 protein.The immunofluorescence results showed that the recombinant hZP2 protein expressed in vitro could not bind to uncapacitated sperm.The results of inhibition experiment showed that the curve of recombinant hZP2 protein was similar to that of uncapacitated sperm before and after binding,and the difference was small.By changing the number of sperm,the concentration of Ca²⁺and the incubation time,there was no correlation between the binding efficiency of uncapacitated sperm and recombinant hZP2 protein.We accurately detected all genotypes in the 3 loci with polymerase chain reaction-restriction fragment length polymorphism?PCR-RFLP?techniques,while we detected no heterozygosity at the rs77853330 locus in NAP5.The similar distributions of genotypic frequency between both groups suggest that the three SNPs investigated in this study are not associated with PD in northern Han Chinese?P>0.05?,and the DP>0.5 of 3 loci,which was a genetic marker with high discriminating ability.Conclusion:1.The recombinant hZP2 protein expressed in eukaryotic system was successfully obtained.2.No recombinant hZP2 protein expressed in eukaryotic system was found to bind to uncapacitated sperm in vitro.3.The 3 SNPs and 1 inDel investigated in DLG2,RBMS3 and NAP5 genes are unlikely to play roles as common risk factors for PD disease in the northern Han Chinese population.In forensic science,it is a genetic marker with high discriminating ability.