| Sub-cellular comparative proteomic research strategies and techniques were applied in this study. Nucleus, mitochondria, lysosome and Golgi body were isolated from the model of mammary gland in dairy cows by ultra-centrifugation technique. Gel profiles of protein expression were analyzed and differential protein spots were screened by PDQest7.4 software. Differentially expressed proteins were analyzed and identified by Mass Spectrometry and biological informatics.Some proteins related to mastitis in mitochondria, nucleus,lysosomes and Golgi body were detected respectively, their functional mechanism were analyzed and discussed in the pathogenesis of mastitis in dairy cows.Mammary gland tissue from Diary cows was subject to diferential centrifugation and four fractions of 1000g, 6000g and 20000g pellets were recovered. Then density gradient centrifugation was applied to the 1000g and 6000g pellets for further preparation of nuclei and mitochondrial, and 20000g pellet for preparation of lysosomes and Golgi body.The purity of nuclei and mitochondrial was increased 6-fold by this preparation.The proteins of nuclus, mitochondrial, lysosomes and Golgi body were separated with two-dimensional gel electrophoresis (2-DE).583,477, 509and 488 protein spots were detected in the four fractions of mitochondria ,nucleus, Golgi body and lysosomes respectively, the total number of protein spots of the four fractions was about 2000, whereas just about 480 spots could be acquired for mammary gland homogenates. Since most of the mitochondrial and nuclear proteins were not able to present themselves in the 2-DE profile of the cell homogenates, more low-abundance proteins were detected through sub-cellular fractionations.Protein changes in the mammary gland between healthy cows and clinical mastitis cow were investigated, the differentially expressed proteins were related to cell energy metabolism, cell migration, molecular chaperone, anticoagulation mechanism, inhibit inflammatory response, cell differentiation and signal transduction,which may be involved in the body defense mechanism against the mammary gland infection process. It has been found that the expression of mitochondrial enzymes such as bovine mitochondrial ATP enzyme-F1, bovine heart cytochrome c oxidase, cytochrome Bc1 complex, acyl-coenzyme A synthetase family of 3, Q-base ribosomal tRNA-transferase and 1 amide acid phosphate ribosyltransferase were up-regulated in infected mammary tissues. The expression of casein proteins (casein proteinα-S2, casein proteinα-s1,β-casein,β-casein A2 mutant protein, casein protein-κ) were down-regulated in infected mammary tissues, which may lose the ability of casein and lead to milk condensation. The expression of Hsc70 was up-regulated dramatically in mammary gland tissue infected with clinical mastitis, and may lead to speed up the energy metabolism and prevent cell from injury. Hsp90 was missed in mastitic cows, which may be related to the repeated occurrence of mastitis, and may serve as new susceptible markers of mastitis and potential protein targets for trentment. Membrane protein V and membrane protein A7 were up-regulated in mastitis cows,which may regulate blood clots and inflammatory process, and be regarded as inflammatory mediated protein and potential protein targets for mastitis treatments.Albumin-related anti-inflammatory proteins was up-regulated. Transporter protein-related substances such as external orifice of membrane channel proteins and apolipoprotein A-I were down-regulated.β-lactoglobulin and thioredoxin were down-regulated; Zinc finger protein was up-regulated,which may be potential biomarkers during mastitis in dairy cows.In conclusion, these results may provide valuable information for the investigation of the host mammary immune system response to defense against pathogens and involving in the pathogenesis of mammary inflammation at sub-cellular level, and finding new diagnostic markers of mastitis and potential protein targets for treatment.
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