The Construction Of Two-dimensional Gel Electrophoresis Experimental System And The Differential Proteomic Analysis In Ovine Follicular Fluid

Over the years, sheep is the main livestock in Xinjiang animal husbandry. In addition to the promotion of scientific feeding technology, improve sheep reproduction ability also is is the main way to promote the development of the sheep industry. Rapid propogation using reproductive technologies ET, IVF of sheep, geting the high quality oocytes is the key of in vitro embryo production technology. Follicular fluid provides a micro environment for the growth and development of oocyte. Proteomics research on follicular fluid, can help us to find out the effect of oocyte development key factor.In this study, the influence two-dimensional electrophoresis(2-DE) factors of ovine follicular fluid protein, including sample preparation, gel concentration, strips, sample volume and staining method,were been optimized. Using the growing follicular fluid samples from ewe and lamb which induced by hormone and from different diameters of follicles proceeding 2-DE followed by tandem mass spectrometry analysis were conducted. The results of this study are as follows:1. Sheep follicular fluid samples to remove high abundance serum albumin by PierceTM Albumin Depletion Kit, 12.5% acrylamide gels and bands Immobiline DryStrip 18 cm, pH3-10 of 250?g protein samples for 2-DE.2. The three groups of different diameter follicular fluid samples(<3mm, 3-5mm, >5mm) were collected for 2-DE analysis. The seventeen different protein spots were identified,and only seven protein spots identity were abtained annotion: tubulin beta-2A chain, clusterin, tropomyosin 3, alpha-1-acid glycoprotein, actinbeta and albumin(of which there are two protein spots was identified as albumin).These proteins highly expressed in < 3mm follicular fluid than the orther two groups.3. The 2-DE and MS proteomic analysis of hormone stimulated lamb and ewe growing follicle fluid proteom. There 16 difference protein spots were identified,and only six protein spots identity were identified: hemoglobin subunit alpha-1/2(two protein spots were identified as the protein), hemoglobin subunit beta-C, protein HP-25 homolog 1, APOA1, HP. The first four were higher in ewe, and the last two were higher in lamb.