Preparation And Application Of Antibody Against Pig Cysteine-rich Secretory Protein 1(CRISP1)

Fertilization is the starting point of mammalian life,including sperm's and egg's identification,fusion and formation of a zygote ?Ltimately.During last two years,with the rapid development of biotechnology,we have more understanding for the fertilization process,with a large number of proteins being shown to participate in the this process,however,the molec?Lar mechanism of the fertilization is still not completely clear.According to current findings,CRISP1 is an important protein involved in fertilization process,and the expression of the protein has been found in the reproductive system of male and female mice.,Some studies have indicated that the protein may be involved in sperm capacitation and fusion processes.However,there has been no relevant study reported concerning CRISP1 proteinin porcine until now.In this study,the prokaryotic expression of porcine CRISP1 protein was used to prepare the polyclonal antibody against porcine CRISP1.The expression of CRISP1 protein in germ cells was tested by immunofluorescence method.In addition,we investigated the role of this protein in fusion process by in vitro fertilization.PET-32 a vector was utilized for the prokaryotic expression system.The c DNA sequence of porcine CRISP1 was amplified through RT-PCR,and then cloned into the PET-32 a vector,named by PET-CRISP1.PET-CRISP1 vector was transformed into E.coli Rosetta and induced to express HIS-CRISP1 fusion protein,which was purified by gel cutting.This purifed HIS-CRISP1 fusion protein was used to immunize the ad?Lt Kunming female mice,which were then injected with S180 cells to obtain the polyclonal antibody against CRISP1.To test this polyclonal antibody,the whole crude protein of porcine heart,testis and epididymis as antigen were used to do Western blot,respectively,the res ?Lt showed that this antibody worked well..In this study,we demonstrated that CRISP1 protein was not only expressed in the Sertoli cells and spermatozoa of testis but also located in oocytes and cum?Lus cells in Sow.In addition,we also detected its location by immunofluorescence before and after capacitation of boar sperm,and we found that before capacitation,CRISP1 was mainly accum?Lated in the head and tail joint part of sperm,while after capacitation,the protein was expressed in the the equatorial plate of sperm head.In vitro fertilization experiments,we added polyclonal antibody(experimental group)and reference ascites(reference group)in semen,but the difference of fertilization rate and splitting rate between the two groups was not significant,but there was a downward trend,indicating that the CRISP1 protein might be involved in the process of fertilization process of oocyte and sperm,such as recognition and fusion etc.