Identification Of The Interaction Between SLLP1 And PDIA3 Of Sheep

To explain fertilization process at molecular level is a big challenge in sexual reproduction studies.Getting over the difficulties in fertilization process is important for fertility and successful in vitro fertilization.In this study,sperm and egg fuse together remarkably and form a zygote,and insufficiency of protein-protein interactions cause undetermined infertility.For the fertilization process to be successful,sperm has several disputes to penetrate the egg.Fertilization involves three main processes:sperm capacitation,sperm and zonapellucida combination and acrosome reaction,and sperm-egg membrane fusion,sllp1 is encoded by SPACA3 at locus 17q11.2.SLLP1 antibody and recombinant protein block in vitro fertilization and sperm egg binding in mice,suggesting that SLLP1 may play an important role in sperm egg interaction.PDIA3,which was also known as ERp57,is a member of protein disulfide isomerase(Protein disulfide isomerase,PDI)family.Previous studies suggest that the PDIA3 may also be involved in sperm swimming and gamete fusion.The interaction between these two proteins is still unknown.Therefore,we cloned sllpl from the testis tissue of male sheep(ovis aries).The full length of sllpl cloned was 566 bp.We construct recombinant vector pET-28a-sllp1 for prokaryotic expression.The recombinant protein SLLP1-HIS is expressed and purified.In this study,yeast two hybrid Recombinant vectors pGAD-sllpl and pGBK-pdia3 were constructed,PDIA3 expressed in Y2H Gold and SLLP1 expressed in Y187,respectively.Blue colonies grow out in the SD/-Trp/-His/-Ade/-Leu/X-a-Gal/AbA medium.,there also grow out the blue colonies.The result indicateds a preliminary interaction between the two proteins,we construct pACT-pdia3 pBIND-sllpI vectores for mammalian two hybrid,themammalian two hybrid results are expressed as mean ± standard deviation(n=3),and single factor analysis of variance is performed.The active-control groupp<0.01,the difference is significant;the experimental group p<0.01,the difference is very significant,indicating that PDIA3 and SLLP1 in 293T cells have strong interaction.The above experimental results indicate that there is direct interaction between PDIA3 and SLLP1.We hypothesized that SLLP1 may be involved in protein phosphorylationduring sperm capacitation.In addition,the use of Swiss model to predict SLLP1 three-dimensional structure model and its sequence analysis data has a high consistency,the three-dimensional structure model is highly reliable,can be used for its functional research.