Preliminary Screening For The Protein Interacting With Rat CRISP2

Sexual reproduction is a very important breeding method among the mammalian.The sperm produced by male animals is combined with the eggs produced by the female to form a fertilized egg,and then the fertilized egg develops into a new individual.In the process of sperm and egg fusion,many interactions between protein molecules,in particular,the interaction between the membrane proteins of sperm and egg have very important significance.So far,although at least three pairs of interacted proteins on sperm and egg have been identified,but only the interaction between the sperm surface protein IZUMO1 and the egg surface protein JUNO is essential to sperm-egg fusion.However,the interaction of IZUMO1 with JUNO alone could not lead to membrane fusion,there should be other sperm and egg proteins involved in sperm egg fusion.The sperm protein CRISP2—a secreted protein that are rich in cysteine,was found to bind to egg surface.Knockout crisp2 could significantly inhibit fertilization,indicated that the CRISP2 may play a important role in sperm egg fusion through a putative receptor on egg surface,but the receptor hasn?t been identified yet until now.This experiment was designed to screen the predicted CRISP2 receptor from rat ovary by yeast two hybrid method.The bait recombinant plasmid pGBK-crisp2 was constructed,and hybridized with rat ovary cDNA library to hook any proteins that interacted with CRISP2.The experiment got three clones which included cDNA fragments of creb3l1,tl0aea56yl02 and chromosome 20,respectively.Among them only creb3l1 was reported as a transmembrane protein,leaving us a clue to further verify whether the CREB3L1 is on the egg surface and can bind to CRISP2.The remained two cDNA did not belong to a known gene.In order to identify the location of CREB3L1 on egg and its interaction with CRISP2,we constructed the recombinant prokaryotic expression vector of pGEX-crisp2 and expressed recombinant fusion protein CRISP2-GST in E.coli BL21(DE3)successfully.By injecting CRISP2-GST into the Kunming white female mice,we obtained the mouse anti rat CRISP2-GST polyclonal antibody,the antibody will be used in yeast two hybrid and indirect immunofluorescence experiments.