Identification And Analysis Of Differentially Expressed Proteins In Sheep Mammary Gland Tissue(Ovis Aries)

Mastitis was one of the most common multiple diseases in livestock husbandry and aquaculture.It would lead to decrease milk production,change of milk composition and physicochemical properties of lactating animal,etc,which would reduce the nutritive value and economic value of dairy products,giving livestock husbandry industry brought to huge economic losses.Therefore,in this study,mammary gland tissue of normal and clinical mastitis sheep as the experimental material,the mammary gland tissues proteins of normal and clinical mastitis sheep were analyzed and identified using two-dimensional electrophoresis,mass spectrometry and bioinformatics technology.Afterwards,four differentially expressed proteins were verified by real-time fluorescence quantitative PCR,Western blot and immunohistochemistry.The main results as follows:1.Establishment and optimization of 2-DE system of sheep mammary gland proteinA set of suitable 2-DE system of sheep mammary gland protein separation was established by comparing and optimizing the four parameters of different loading amount,different focusing time,different separation gel and different size pH strips.The results showed that the sheep mammary gland protein could be well separated using trichloroacetic acid(TCA)/acetone precipitation method,loading 600 μg,focusing time 90000 Vh or higher,17 cm pH 3-10 strips and 12% of the separation gel could be better separate the sheep mammary gland protein.2.Normal and clinical mastitis sheep mammary gland proteomics researchBased on the optimization and establishment of sheep mammary glands protein 2-DE system,two-dimensional gel electrophoresis and mass spectrometry identification techniques were used to identify and separate the mammary glands differential proteins of normal and clinical mastitis sheep and done bioinformatics analysis.The results showed that 63 differentially expressed protein spots were detected from gel pattern of the clinical mastitis sheep mammary gland.37 protein spots were identified by the mass spectrometry and database search,a total of 30 proteins were found(up-regulation of 18 proteins and down-regulation of 12 proteins).GO clustering analysis found that these differential proteins were mainly involved in physiological processes such as cell,metabolism,immunity,signaltransduction,and biological regulation and were mainly involved molecular functions such as binding,molecular structure activity,transport activity,and catalytic activity.KEGG enrichment analysis found that these differentially expressed proteins were mainly involved in five major signaling pathways such as metabolism,genetic information processing,environmental information processing,cellular processes,and biological systems.Protein interaction network analysis revealed 14 differentially expressed proteins had significant interactions relationship.3.Verification of four differentially expressed proteinsThe differential proteins SOD2,ANXA2,ERp29 and KRT10 were detectd in normal and clinical mastitis sheep mammary gland by Real-time quantitative PCR and Western blot.The results showed that the expression trend of SOD2,ERp29,and KRT10 proteins at the level of transcription were consistent with that of 2-DE result.The expression trend of ANXA2 protein was opposite to that of 2-DE result.The expression trend of SOD2,ANXA2,ERp29 and KRT10 proteins at the protein level were consistent with that of 2-DE result.Immunohistochemical methods revealed that SOD2,ANXA2,ERp29 and KRT10 proteins were expressed in both normal and clinical mastitis sheep mammary glands and were localized in mammary epithelial cells.