The Differential Proteomic Study Of Hu Sheep Milk In Different Lactation Period

The current thesis carried out a proteomic analysis of skim milk and whey protein after 3,000×g centrifugation and 100,000×g ultracentrifugation, respectively, from six time points during postpartum period(1 d, 3 d, 7 d, 14 d, 28 d and 56 d). The main results were as follows:(1) Analysis of skim milk protein in different lactation of Hu sheepThe proteomic feature of skim milk was analyzed using 2-DE technology. A total of 44 differentially expressed protein spots(DEPS) were found in skim milk from 6 time points during lactation period. Taking the similar research on Sarda sheep skim milk as a reference, we found these skim milk proteins in Hu sheep were alpha lactalbumin(α-La), β-Lg, αS1-CN, αS2-CN, beta-casein(β-CN), kappa-casein(κ-CN), albumin(Alb) and Ig heavy chain precursor. The DEPS between 1d and 3, 7, 14, 28 and 56 d were 38, 35, 34, 38 and 36, respectively.(2) Analysis of whey milk protein in different lactation of Hu sheepThe whey protein of all raw skim milk was obtained after ultracentrifugation. Then, 2-DE and MALDI TOF/TOF mass spectrometry were applied to study the proteomic features of whey protein at 6 different lactation time points of Hu sheep. As a result, a total of 41 DEPS were found, which identified 15 proteins. Of them, six proteins were directly match to sheep proteomic database, including κ-CN(CSN1), αS1-CN(CSN1S1), clusterin(CLU), alpha-1-antitrypsin transcript variant 1(A1ATV1), airway lactoperoxidase(ALPO) and kinesin-like protein(KLP); and the remaining 9 proteins were highly matched to homologies in vertebrate proteomic database, including immunoglobulin s25705 Ig mu chain(Ig μ), complement C3, polymeric immunoglobulin receptor isoform x2(PIGR2), immunoglobulin heavy chain constant region of tetrameric 1a membrane form(Ig CH1a), nucleobindin-1 isoform x1(NUCB1), vitamin d-binding protein(BDP), keratin(KRT), glycosylation-dependent cell adhesion molecule 1(Gly CAM1) and cytoplasmic 2isoform x1 of actin(ACTG1).The DEPS of whey protein at 3, 7, 14, 28 and 56 d with 1 d were 30, 29, 39, 20 and 31, respectively. Based on the changing amount of protein abundance, CLU showed a high expression level at the first 7 d of lactation, while revealed a dramatical decrease at 14 d, and was even in absence of expression at 28 d and 56 d. The abundance of A1AT1 was in the highest level at 14 d.The content of ALPO was higher at early stage of lactation, and rapidly decreased at 7 d, even until to 0 at 14 d, then increased at 28 d, while decreased to 0 at 56 d. The abundance of kinesin-like protein was higher only at 7 and 14 d. The abundance of complement C3 was very high at 1 d, and then showed an irregular changing tendency at remaining time points. The BDP revealed high expression at 1, 7 and 28 d, while low expression at 3, 14 and 56 d. Gly CAM1 was abundant at 7 d and 14 d. The abundance of actin was high at 1 d, and then decreased, down to 0 at 28 d and 56 d.(3) Analysis of proteomic difference of Hu sheep milk in different lactationBased on i TRAQ technology, a total of 310 proteins were identified in Hu sheep milk whey at 6 time points during lactation. Of which, 111 proteins were directly matched to sheep proteomic database, and 199 proteins were matched to vertebrate proteomic database. In addition, 121 proteins show differential expression among different time points, and their changing tendency was directly shown in heatmap. Compared with 1 d, the DEPS at 3 d, 7 d, 14 d, 28 d and 56 d were 30, 42, 63, 80 and 85, respectively.According to the molecular function of GO functional annotation, the 121 DEPS identified at different lactation period could be classified into 9 groups, including structural molecule activity, transcription factor activity, antioxidant activity, molecular transducer activity, binding, transporter activity, catalytic activity, chemoattractant activity and molecular function regulator.The DEPS of 1 d vs 3 d contained the most number of molecular functions. In the molecular function of DEPS of 3 d vs 7 d, transcription factor activity, antioxidant activity was lost. Structural molecule activity, transcription factor activity, antioxidant activity and chemoattractant activity were lost in 7 d vs 14 d, 14 d vs 28 d, 28 d vs 56 d.According to the cellular location of GO functional annotation, the DEPS could be classified into 9 groups: organelle, cell, extracellular region, membrane-enclosed lumen, synapse, cell junction, macromolecular complex, extracellular matrix and membrane. The DEPS of 1 d vs 3 d contained the most number of cellular locations. In the cellular locations of DEPS of 3 d vs 7 d, synapse was lost. In the DEPS of 7 d vs 14 d, synapse and cell junction were lost. The classification of DEPS in 14 d vs 28 d was at least. However, the classification of DEPS in 28 d vs 56 d increased, namely, macromolecular complex and membrane-enclosed lumen.According to the biological process of GO functional annotation, the DEPS could be classified into 17 groups, developmental process, multicellular organismal process, cellular process, single-organism process, biological adhesion, metabolic process, localization, cellular component organization or biogenesis, immune system, signaling, behavior, response to stimulus, growth, locomotion, biological regulation, multi-organism process and rhythmic process. Through the different milking period, we found 6 kinds of biological process showing great changes, namely, biological adhesion, behavior, growth, locomotion, multi-organism process and rhythmic process.In addition, the above 121 DEPS mainly involved in immune, metabology and signal transduction pathway based on metabolic pathway annotation analysis.