The Proteomics Analysis Of Milk Fat Globule Membrane Proteins From Transgenic Cloned Cattle And The Milk Proteins From Chinese Holstein, America Holstein, Chinese Buffalo And Jersey Cattle

The milk fat globule membrane (MFGM) proteins has many important physiological functions. And the MFGM proteins come from endoplasmic reticulum of the mammary gland epithelial cell and. The research of the transgenic cloned MFGM proteins not only evaluation on the safety of transgenic cloned cow’s milk protein composition, but also can reflect the breast health of the milking cows. In this study, we compared the colostrum and mature MFGM protein expression profile of three cloned transgenic bovine lines, i.e., expressing recombinant human a-lactalbumin (TC-LA), lactoferrin (TC-LF), and lysozyme (TC-LZ), with that of cloned non-transgenic and conventionally bred animal controls. We identified1225proteins in milk MFGM,166of which were specifically expressed only in the TC-LA group,265only in the TC-LF group, and184only in the TC-LZ group. There were43proteins expressed only in the transgenic cloned animals, but the concentrations of these proteins were below the detection limit of silver staining. Functional analyses also showed that these43proteins had no obvious influence on the bovine mammary gland. Quantitative comparison revealed MFGM proteins that were up-or down-regulated more than twofold in each group compared to conventionally bred animals:126in colostrum and77in mature milk of the TC-LA group;157in colostrum and222in mature milk of the TC-LF group;49in colostrum and98in mature milk of the TC-LZ group; and98in colostrum and132in mature milk in the cloned control group. These up-and down-regulated proteins in the transgenic animals were not associated with a particular biological function or pathway. This study demonstrated that the expression profiles of MFGM proteins in transgenic cloned animals were similar to those of cloned and conventionally bred animals, it appears that expression of certain exogenous proteins has no general deleterious effects on the bovine mammary gland.Totally1,148kinds of proteins has been identified in the whey and fat globule membrane proteins both in colostrum and regular milk of above four kinds of cattle, using high-performance liquid chromatography combined with mass spectrometry methods. Comparing all the proteins that expressed in Jerseys Cattle or Chinese buffalo with the Holstein cattle, there are132kinds of proteins that are specifically expressed in Chinese buffalo, while131different proteins are specifically expressed in Jerseys Cattle. We found out a large number of proteins related to the synthesis and secretion of milk fat by analyzing these specifically expressed proteins.We also detected the expression level of these proteins by iTRAQ quantitative proteomics mass spectrometry. We did clustering and principal component analysis on168different kinds of proteins that are quantitatively determined in colostrum and regular whey milk, and further found the correlation of expression differences between U.S. Holsteins, Jerseys or Chinese Buffalo with Chinese Holstein cattle. A total number of710kinds of protein in 8samples of milk fat globule membrane proteins are given the quantitative results using iTRAQ experiment.Through the identification and functional analysis of differentially expressed proteins, we found a number of proteins that are related to the lipid metabolism, inflammation and other disease. The genes of these proteins may be considered to be the good candidates to breed the GM cows that have the distinct characters, such as improved milk quality or be resistant to cow disease.A set of comprehensive proteomic analysis for dairy products have been established, including sample collecting, pre-processing, high performance liquid chromatography (HPLC) combined mass spectrometry (LC-MS/MS) analysis and post-sample data processing procedure. We also completed the whey protein expression profile analysis for the milk of one cloned cattle, three kinds of transgenic cloned milk. These analyses will provide the scientific data to the safety evaluation of GM milk products, as well as provides the technical tools to the safety evaluation for GM animals.