小鼠E型肉毒毒素中毒血清蛋白差异分析

Differential analysis of serum proteins in mouse suffered from botulinum toxin E poisoning

目的以小鼠为动物模型研究E型肉毒毒素中毒血清蛋白变化,寻找中毒相关蛋白,为肉毒中毒早期诊断及预后寻找新的生物标记分子。方法经腹腔注射昆明系近交小鼠E型肉毒毒素0.75 LD50,分别于24、48、72 h后眼眶采血,分离血清,SDS-PAGE结合2-DE-SDS-PAGE分离血清蛋白,PDQUest 8.0软件对凝胶进行背景消除和点的匹配,离子阱质谱检测差异蛋白点。结果SDS-PAGE条带显示,中毒24 h血清蛋白变化最明显,正常血清和中毒24 h血清2-DE图谱找到20个差异蛋白点,其中14个只存在小鼠正常血清中,3个只存在肉毒中毒血清中,3个在肉毒中毒呈现低量表达。质谱检测出晶体蛋白家族,只在正常小鼠血液中表达;急性期蛋白在E型肉毒毒素中毒24 h出现明显变化,其中结合珠蛋白在中毒血清中低表达,而血清淀粉样蛋白A高表达。结论这些鉴定出的蛋白涉及机体能量代谢、肌肉收缩功能、细胞应激反应、转录、翻译以及细胞增殖的改变,尤其是结合珠蛋白和血浆淀粉样蛋白可初步作为潜在的诊断标记分子。

Objective To investigate the change of serum proteins of botulinum toxin E using mouse model and find poisoning-related proteins to provide novel biomarker molecules for early diagnosis and prognosis of botulinum toxin poisoning.Methods Inbred Kunming mice were injected i.p.with botulinum toxin E at a dosage of 0.75 LD50,of which the serum samples were collected 24,48 and 72 h after injection.Serum proteins were separated by SDS-PAGE coupled with 2-DE-SDS-PAGE,while the gel background was eliminated and the protein spots were matched by using PDQUest8.0 image analysis software.The differential protein spots were identified by high performance liquid chromatography tandem ion trap mass spectrometry.Results SDS-PAGE showed that serum proteins changed significantly 24 h after poisoning.Twenty differential protein spots were distinguished on the 2-DE profiles of normal serum and the serum 24 h after poisoning,of which 14 protein spots existed only in the normal serum while 3 only in the serum after poisoning,and3 were low expressed in the serum after poisoning.Mass spectrometry showed that crystal proteins family was only expressed in normal serum.Acute phase proteins showed an obvious change in the serum 24 h after poisoning,of which haptoglobin was low expressed while serum amyloid A(SAA)protein was high expressed.Conclusion The identified proteins involved in alterations in energy metabolism,muscle contractile function,cellular stress response,transcription,translation,and cell proliferation.Especially,haptoglobin and SAA may be regarded as potential marker molecules for diagnosis.