Isolation, Identification And Bioinformatics Of Differential Expression Proteins In Plasma Of Cows Affected By Milk Fever

Background: Hypocalcemia in dairy cows is divided into clinical type, named as milk fever,and subclinical type. Milk fever is an important nutritional and metabolic disease characterized bythe plasma Ca concentration under the normal level, paralysis and even coma around calving.Milk fever may induce many perinatal conditions, such as ketosis, endometritis, abomasumsdisplacement, and even lead to die. In past researches, the exploration for pathogenesis of milkfever mainly focuses on physiology, biochemistry and pathology. However, there is little reportabout the proteomics of milk fever. Therefore, it has an important significance in this study toreveal the global change of life functions during the development of milk fever, enrich anddevelop the its pathogenesis.Objective: To find the differential expressed proteins among the milk fever cows (MF),subclinical hypocalcemia (SH) cows and healthy (C) cows by fluorescence two-dimensionaldifferential gel electrophoresis (2D-DIGE) combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and to explore effect of theseproteins on milk fever in dairy cows.Methods: Twenty seven cows were divided into three groups in this study, which were MF,SH and C groups, and nine cows in each group. In same group, three plasma samples were mixedin same volume as a sample, and then was labeled with fluorescence dyes after removal of highabundance proteins, such as serum albumin and IgG. After sample proteins were separated by2D-DIGE, the different proteins were collected from gels using the DeCyDer software on thebasis of difference mutiple>1.5, and P<0.05. These proteins were identified by MALDI-TOF-MSand the search of NCBI database, and then were analyzed online through bioinformatics.Results: Electrophoretogram of plasma samples from cows with MF, SH and C groups wereobtained by2D-DIGE,13differential expressed proteins were separated and identified byMALDI-TOF-MS and the search of NCBI database, and results showed that①compare with theC group,3upregulated proteins in MF group were amine oxidase, copper containing3(AOCC),immunoglobulin M (IgM) and unnamed protein, and7down-regulated in MF group were albumin(ALB), alpha1-antichymotrypsin (ACT), Fibrinogen (FG), complement component C3d (C3d),fibrinogen A-alpha chain (FGA), fibrinogen beta chain (FGB) and fibrinogen, gamma chain(FGG);②compare with the C group, there was no up-regulated protein in SH group, and6down-regulated proteins were serpin peptidase inhibitor (SERPIN), alpha-2-macroglobulin (A2M),Complement component4A (C4A), IgM, FG and FGB；③compare with the SH group, there isonly1up-regulated SERPIN, and7down-regulated proteins were ALB, ACT, FX, FG, FGA, FGBand FGG. Eight proteins searched and analyzed in the online analysis of Networks, GO, andPathway, were A2M, FGA, FGB, C(C4A), ALB, IgM, FX and SERPIN. It suggest that theseproteins were maybe related to the milk fever and hypocalcaemia.Conclusions: In this study, we first explore plasma proteomics of MF, SH and C cows,separate and identify the13differential expressed proteins from MF and SH cows using2D-DIGE/MALDI-TOF-MS, and then discuss relationship between them and milk fever, however, it needto verify these proteins and their relationship with milk fever in the future, and offer a newstrategy to interpret mechanism of milk fever and to assess state of an illness and effect ofprevention and cure in future.