应用蛋白质组学筛选能量负平衡卵巢静止奶牛生物标记物

Application of proteomics in screening biomarker for energy negative balancing in cows with inactive ovary

目的运用蛋白质组学技术筛选能量负平衡卵巢静止奶牛的生物标记物,建立早期诊断标准,并探讨其发病机制。方法应用荧光差异双向凝胶电泳(fluorescence two-dimensional differential gel electrophoresis,2D-DIGE)和基质辅助激光解析离子化飞行时间质谱(matrix-assisted laser desorption/lonization time of flight mass spectrometry,MALDI-TOF-MS),鉴定能量正平衡发情奶牛和能量负平衡卵巢静止奶牛的差异蛋白质。采用生物信息学、蛋白印迹和受试者工作特征(receiver operating characteristic,ROC)曲线分析差异蛋白质,并确定用于早期诊断卵巢静止的生物标志物。结果两组奶牛筛选出12种差异表达蛋白,这些差异蛋白主要参与脂类代谢、维生素代谢、补体凝血级联及过氧化物酶增殖物激活受体(peroxisome proliferator activated receptor-α,PPAR)信号通路,其中α-1-微球蛋白/bikunin前体(alpha-1-microglobulin/bikunin precursor,AMBP)可作为卵巢静止早期诊断的标志物,预警值为4 346.75 ng/L。结论应用蛋白组学筛选出能量负平衡导致奶牛卵巢静止的12种差异表达蛋白,确定了用于早期诊断卵巢静止的生物标志物,为卵巢静止的临床诊断和发病机制奠定了基础。

Objective To use proteomics techniques to screen biomarkers which negatively balance the energy in cows with inactive ovary,establish early diagnostic criteria and investigate the mechanism of pathogenesis.Methods Fluorescence two-dimensional differential gel electrophoresis(2 D-DIGE)and matrix-assisted laser desorption/ionization time-offlight mass spectrometry(MALDI-TOF-MS)were used to identify differentially expressed proteins in energy-balanced estrous cows and energy-negative balanced cows with inactive ovary.Bioinformatics,Western blot and receiver operating characteristic(ROC)curve analysis were used to analyze differential proteins and determine the biomarkers for early diagnosis of inactive ovary.Results Twelve kinds of plasma differentially expressed proteins were screened in the two groups of cows.The proteins were mainly related to lipid metabolism,vitamin digestion and absorption,complement and coagulation cascade as well as PPAR signaling pathway,among which alpha-1-microglobulin/bikunin precursor(AMBP)might be used for markers for early diagnosis of inactive ovary,with an early-warning value of 4 346.75 ng/L.Conclusion Twelve kinds of differentially expressed proteins resulting in inactive ovary by negative energy balance in dairy cows were screened by proteomics,which was used as a biomarker for the early diagnosis of inactive ovary.It laid a foundation of clinical diagnosis and research on pathogenesis of inactive ovary.