Preparation Of Polyclonal Antibody Against Sheep SAS1B Gene And Its Effect On Fertilization

Mammalian fertilization is a considerably sophisticated process and sperm crossing the oocyte zona pellucida to achieve sperm-egg fusion figures prominently in the entire process.However,up to now,the molecules involved in this process and their roles have been poorly understood.SAS1 B is a newly discovered novel egg membrane protein associated with sperm-egg fusion,which is specifically expressed on the oocyte membrane.SAS1 B has the ability to recognize and bind to the SLLP1 protein in the equatorial region of the sperm acrosome to achieve sperm-egg fusion during fertilization.Sheep is an important agricultural economic animal,and its fertility level directly affects the economic benefits of sheep breeding.The success of sheep sperm-egg fusion is directly related to the lambing rate of sheep,which is of great significance to improve the reproductive efficiency of sheep.In recent years,SAS1 B expression and its related effects on fertilization were mainly studied in mice,which were found to be related to follicular development and fertilization.However,there was no reports of studies on the fertilization process in sheep.To investigate the effect of SAS1 B on sheep fertilization,this study first cloned the full length sequence of CDS region of SAS1 B gene from sheep ovary,constructed prokaryotic expression vector,expressed and purified its recombinant protein,immunized Japanese white rabbits with purified recombinant protein to obtain sheep SAS1 B polyclonal antibody,and detected the expression of SAS1 B in sheep ovaries.Finally,the effect of SAS1 B on sheep fertilization was preliminarily explored by recombinant proteins and polyclonal antibodies,which laid a foundation for the study of SAS1 B protein’s function and its mechanism during sheep fertilization.The research results are as follows:1.The full length sequence of CDS region of sheep SAS1 B gene was cloned with1527 bp,encoding a total of 508 amino acids.The amino acid sequence analysis showed that SAS1 B protein was predicted to be a hydrophilic protein with high evolutionary conservatism.2.Codon optimization was performed on the CDS region of sheep SAS1 B gene with an optimized sequence length of 1524 bp.The recombinant vector p ET-28a(+)-SAS1 B was constructed.The vector band of 5369 bp and the target band of 1524 bp were identified by double enzyme digestion.SAS1 B recombinant protein with a size of 48 k D was induced.At0.1 mmol/L IPTG and 16℃for 18 h,a large amount of induced SAS1 B soluble recombinant protein was obtained.The purified sheep SAS1 B recombinant protein was obtained by nickel column affinity purification.3.A rabbit polyclonal antibody against sheep SAS1 B was prepared.The titer of the antibody was 1:25600 by indirect ELISA,and the antibody exhibited favourable reactivity with sheep SAS1 B protein by Western blot.Immunohistochemical staining of sheep ovary with antibodies revealed that SAS1 B was expressed in all levels of follicles and granulosa cells of sheep ovariy.With the follicular development,SAS1 B expression increased significantly in secondary follicles(P < 0.05)and reached the highest level in mature follicles;Subsequent immunofluorescence staining of stage MⅡ oocytes and fertilized eggs showed that SAS1 B was located in the microvilli region on the oocyte membrane.4.Addition of sheep SAS1 B protein at a concentration exceeding 25 μg/m L sperm capacitation could promote sperm capacitation and accelerate acrosome reaction.However,the fertilization rate significantly decreased(P < 0.05).Incubation of mature sheep oocytes with sheep SAS1 B polyclonal antibody could significantly inhibit fertilization and significantly reduce the fertilization rate of oocytes(P < 0.05).In summary,this study successfully cloned the CDS region of sheep SAS1 B gene,purified the recombinant protein of sheep SAS1 B,and prepared a polyclonal antibody against sheep SAS1 B.Using this antibody,SAS1 B gene expression was detected to be the highest in sheep mature follicles,and SAS1 B expression was also found in granulosa cells.In sheep MⅡ oocytes and fertilized eggs,SAS1 B was located in the microvilli region on the oocyte membrane,and promoted oocyte development.Sperm capacitation and in vitro fertilization experiments confirmed that SAS1 B protein could promote sperm capacitation and accelerates acrosomal reaction of sperm,which played an extremely important role in the fertilization process of sheep.Moreover,the SAS1 B protein may be a conservative protein that ensures lineage continuation during biological evolution.