排黏大熊猫的血液蛋白质组学研究及其生物信息分析

doi:10.16431/j.cnki.1671-7236.2025.05.002

摘要/Abstract

摘要： 【目的】探讨圈养大熊猫在排黏期与非排黏期血清蛋白质组学特征，以揭示排黏现象的分子机制，并为圈养大熊猫的健康管理提供科学支持。【方法】以8只圈养大熊猫为研究对象，每只大熊猫在排黏期和非排黏期各采集一次血液样本，将样本分为排黏期组(NY)和非排黏期组(Con)。排黏期组在大熊猫排黏后2 h内完成血液采集，非排黏期组血液采集与排黏期相隔至少30 d。提取血液蛋白后进行酶解，获取的肽段经过脱盐和定量处理。采用数据独立采集(data-independent acquisition，DIA)技术进行液相色谱-质谱联用(liquid chromatography-mass spectrometry，LC-MS/MS)分析，数据导入Spectronaut^TM软件，进行分析。检测数据在美吉云平台分析，差异蛋白筛选标准为P＜0.05，且差异倍数(FoldChange，FC)＞1.2或＜0.8。用生物信息数据库对差异蛋白进行聚类分析、GO功能富集分析，对差异蛋白涉及的代谢通路进行富集分析，差异蛋白之间进行蛋白互作分析；采用平行反应监测(parallel reaction monitoring，PRM)验证蛋白数据，将目标蛋白中肽段的定量值根据标肽进行归一化，统计检验的阈值设定为P＜0.05，并与PRM验证结果进行对比。【结果】在大熊猫血液样品中共鉴定出273种蛋白，其中258种为共有蛋白，12种为排黏期特有蛋白，3种为非排黏期特有蛋白。差异表达蛋白分析显示，排黏期组和非排黏期组共筛选出25种差异表达蛋白，其中7种蛋白表达上调、18种蛋白表达下调。差异蛋白主要参与细胞生长、免疫应答及炎症反应等生物过程，且主要富集于Rap1信号通路、雌激素(Estrogen)信号通路、磷脂酰肌醇3-激酶-蛋白激酶B(PI3K-Akt)信号通路和松弛素(Relaxin)信号通路。蛋白互作分析筛选出A0A7N5KNP1(FBLN5)、D2GWB9(THBS1)、D2HUL0(SERPIND1)和D2HHD2(ITGA2)等核心蛋白在排黏期显著变化。【结论】排黏大熊猫与非排黏熊猫血清中发现25种差异表达蛋白。这些蛋白主要富集于PI3K-Akt、Rap1、Estrogen和Relaxin信号通路，其中核心蛋白A0A7N5KNP1(FBLN5)、D2GWB9(THBS1)、D2HUL0(SERPIND1)和D2HHD2(ITGA2)在大熊猫排黏现象中发挥重要作用。

关键词: 大熊猫; 血清蛋白质组; 排黏; 非数据依赖采集(DIA); 平行反应监测(PRM)

Abstract: 【Objective】 The proteomic characteristics of serum in captive giant pandas during mucus-excreting and non-mucus-excreting periods were investigated to elucidate the molecular mechanisms underlying mucus excretion and provide scientific support for the health management of captive giant pandas.【Method】 Eight captive giant pandas were selected for this study,with one blood sample collected from each panda during both the mucus-excreting period and the non-mucus-excreting period.The samples were then divided into the mucus-excreting period group (NY) and the non-mucus-excreting period group (Con).Blood was collected from the mucus-excreting group within 2 h after mucus excretion,while for the non-excreting group,blood collection occurred at least 30 days after the last mucus-excreting period.After blood proteins were extracted and digested,the resulting peptides were desalted and quantified.Data-independent acquisition (DIA) was used for liquid chromatography-mass spectrometry (LC-MS/MS) analysis,and the data were processed using Spectronaut^TM software.Data analysis was conducted on the Majorbio Cloud Platform,with criteria for screening differentially expressed proteins set at P＜0.05 and a FoldChange (FC)＞1.2 or<0.8.These proteins were further analyzed through clustering,GO functional enrichment analysis,metabolic pathway enrichment analysis,and protein interaction analysis with bioinformatics databases.Protein data were validated by parallel reaction monitoring (PRM),with peptide quantification values normalized according to reference peptides.Statistical significance was set at P＜0.05,and DIA results were compared with PRM validation outcomes.【Result】 A total of 273 proteins were identified in the blood samples of giant pandas，with 258 proteins shared between groups，12 proteins unique to the mucus-excreting period，and 3 proteins unique to the non-mucus-excreting period.Analysis of differential protein expression revealed 25 differentially expressed proteins between the mucus-excreting and non-mucus-excreting groups，including 7 up-regulated proteins and 18 down-regulated proteins.The differentially expressed proteins were found to be primarily involved in biological processes such as cell growth，immune response and inflammatory response，with significant enrichment observed in the Rap1，Estrogen,phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt),and Relaxin signaling pathways.Through protein interaction analysis，A0A7N5KNP1(FBLN5)，D2GWB9(THBS1)，D2HUL0(SERPIND1) and D2HHD2(ITGA2) were identified as key proteins，showing significant changes during the mucus-excreting period.【Conclusion】 Twenty-five differentially expressed proteins were identified in the serum of giant pandas during mucus excretion compared to the non-mucus-excreting period.These proteins were primarily enriched in the PI3K-Akt，Rap1，Estrogen and Relaxin signaling pathways，with core proteins A0A7N5KNP1(FBLN5),D2GWB9(THBS1),D2HUL0(SERPIND1) and D2HHD2(ITGA2) found to play important roles in the mucus excretion phenomenon in giant pandas.

Key words: giant panda; serum proteome; mucus-excreting; data-independent acquisition (DIA); parallel reaction monitoring

中图分类号:

S858.9

夏茂华, 李祥翔, 刘金鹏, 国欣欣, 徐海泓, 刘彦晖, 戴春阔, 李曦, 王运盛. 排黏大熊猫的血液蛋白质组学研究及其生物信息分析[J]. 中国畜牧兽医, 2025, 52(5): 1955-1965.

XIA Maohua, LI Xiangxiang, LIU Jinpeng, GUO Xinxin, XU Haihong, LIU Yanhui, DAI Chunkuo, LI Xi, WANG Yunsheng. The Blood Proteomics Study and Bioinformatics Analysis of Mucus-Excreting Giant Pandas[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(5): 1955-1965.

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