| Objective:One of the most common biological samples in forensic physical evidence is the mixed stain of semen and vaginal fluid.It is difficult to separate sperm from the mixed stain in current sexual crimes.There are various methods for detecting mixed semen and vaginal fluid components and detecting semen-specific components.However,due to the number of female components such as vaginal epithelial cells more than that of sperm,it is difficult to obtain sperm.Or even if obtained,the sperm is incomplete or not a single component.These make it difficult to extract sperm DNA,and it is even more unable to conduct personal identification.Zona pellucida(ZP)is a translucent matrix of glycoprotein encapsulated around mammalian oocyte,which plays an important role in fertilization.In humans,ZP consists of four glycoproteins: ZP1,ZP2,ZP3 and ZP4.Among them,human ZP3 is the primary receptor for sperm-egg recognition and binding.During fertilization,it binds to sperm and induces a series of sperm reactions,including sperm membrane activation,tyrosine phosphorylation and acrosome reaction.It plays an important role in sperm-egg binding,acrosome reaction,preventing polysperm entry into eggs,and protecting eggs and preimplantation embryos.The functional peptide segment of hZP3,214-305 aa,(named TrZP3)is the key peptide segment to induce sperm acrosome reaction.It has been proved that recombinant hZP3 and TrZP3 are similar to natural ZP3 in structure and function,and can bind to sperm in vitro.More concerned with research in the field of reproduction is the capacitated sperm,in vitro binding of spermegg cells or sperm-ZP.However,in the field of forensic physical evidence,most of the sperm in mixed stain are non-capacitated,so the extraction of the non-capacitated sperm is a key issue in this study.In the past,the binding of recombinant hZP3 and TrZP3 to non-capacitated sperm in vitro has not been reported.In this study,instantaneous expression recombinant hZP3 and stable expression function truncated ZP3 were used to explore the in vitro combination with non-capacitated sperm,providing a practical method and theoretical basis for sperm extraction in forensic sex crimes.The DNA genetic markers used in forensic physical evidence in personal identification and paternity testing have undergone several generations of genetic markers,including variable number of tandem repeat(VNTR),short tandem repeat(STR),single nucleotide polymorphism(SNP),after those markers,Insertion/Deletion(InDel)has been paid more and more attention in the field of forensic medicine in recent years.InDel has the characteristics of low mutation rate,good stability,high polymorphism and short PCR fragments,which can make up for the application defects of STR and SNP in these areas.There are reports at home and abroad that have developed the InDel detection system kit.However,due to the differences in genetic structure between different populations in different regions,the detection rate of the kit is low.Therefore,it is necessary to invent the suitable InDel kit and apply it to judicial identification in view of the genetic structure characteristics of northern Chinese population.In this study,three autosomal InDel genes,including ACE,DJ-1 and GIGYF2,were selected and their forensic parameters were calculated,which provided effective data for the establishment of an InDel detection system for Han people in northern China,and reliable basis for individual identification and paternity identification using genetic methods at the genetic level in forensic medicine in the future.Parkinson’s disease(PD)is a common neurodegenerative disease.Some genetic and environmental factors have been proved to cause PD,but the etiology is still unclear.Some studies have revealed the relationship between some candidate genetic markers and PD in different populations,but there are few reports about the relationship between InDel and PD.Studies have shown that these three InDel loci may be associated with the risk of PD,so we also analyzed the correlation between the InDel polymorphism of these three loci and PD to provide effective data for the pathogenesis of PD.Methods:1.In this study,the eukaryotic cell line HEK293 was selected as the object.The recombinant plasmid pCDNA-3.1(+)-ZP3-6His was transfected instantaneously,and the protein expression was detected by Western blotting.2.The fusion protein hZP3-6His was purified by Ni-NTA affinity chromatography.3.The purified fusion protein hZP3-6His was incubated with non-capacitated sperm in vitro.The binding effect was detected by immunofluorescence and inhibition experiments.4.Eukaryotic cell line CHO-K1 was selected as the object to construct a CHO cell line stably expressing EGFP-TrZP3-6His fusion protein.The expression of EGFP-TrZP3-6His fusion protein was detected by Western blotting.5.The morphology and fluorescence intensity of stable cell lines were observed by confocal laser microscopy within 48 h.6.The expression of fusion protein EGFP-TrZP3-6His at cell level was detected by indirect cellular immunofluorescence assay.7.The fusion protein EGFP-TrZP3-6His was purified by Ni-NTA affinity chromatography.8.The dose-dependent and timedependent binding of purified fusion protein EGFP-TrZP3-6His to non-capacitated sperm in vitro was detected by inhibition test.9.Phenol-chloroform method was used to extract genomic DNA from PD cases and control groups of Han nationality in northern China.10.Designing the specific primers of these three InDel loci for PCR and electrophoresis.11.Statistical genotype and gene frequency distribution of InDel locus,calculation of forensic parameters and analysis of the correlation with the risk of PD.Results: 1.The fusion protein hZP3-6His was successfully expressed in the eukaryotic cell line HEK293.2.The fusion protein hZP3-6His was purified by affinity chromatography.3.The in vitro binding of purified fusion protein hZP3-6His with noncapacitated sperm was not detected in immunofluorescence assay,but the inhibition test showed a certain degree of difference.4.A CHO cell line stably expressing ZP3(214-305aa)was successfully constructed.5.The fusion protein EGFP-TrZP3-6His was purified by affinity chromatography.6.The dose-dependent and time-dependent binding of purified fusion protein EGFP-TrZP3-6His to non-capacitated sperm in vitro was not detected by inhibition assay.7.There were significant differences in allele X compared with allele 5(OR = 1.378,95% CI = 1.112-1.708,P = 0.003)and genotype 5 / X + X / X compared with genotype 5 / 5(OR = 1.681,95% CI = 1.174-2.407,P = 0.004)in GIGYF2 InDel locus;however,there were no significant differences in ACE and DJ-1 InDel.8.There was no statistical significance between gender and risk of PD after stratification.9.In ACE and GIGYF2 InDel loci DP > 0.5,and it is a genetic marker with high discriminatory ability.Conclusion : 1.The fusion protein hZP3-6His and EGFP-TrZP3-6His were successfully expressed and purified.2.No fusion protein hZP3-6His or fusion protein EGFP-TrZP3-6His was found to bind to non-capacitated sperm in vitro.3.The GIGYF2 InDel may be related to the increased risk of PD in northern China.4.ACE and GIGYF2 InDel loci can be used in forensic personal identification and paternity testing. |
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