慢性弓形虫感染大鼠脊髓组织蛋白质的双向电泳分析和质谱鉴定

Two-dimensional gel electrophoresis and mass spectrometric identification of the proteome of Toxoplasma gondii in the spinal cord of infected rats

目的观察慢性弓形虫感染大鼠脊髓蛋白质组的变化与差异表达,为探讨慢性弓形虫感染对脊髓损伤的分子机制奠定基础。方法将6只SD雄性大鼠随机分为对照组和感染组,每组3只。感染组每鼠腹腔感染纯化的RH株弓形虫速殖子107/ml×2ml,对照组每鼠腹腔注射灭菌生理盐水2ml。大鼠感染弓形虫10周后解剖,采用双向凝胶电泳分离脊髓总蛋白,经考马斯亮蓝染色后用Image Master图象分析系统分析,从胶中选取分离蛋白质点,通过基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)分析获取肽质量指纹图谱(PMF),采用Mascot软件搜索MSDP、SWISS2PROT数据库鉴定蛋白质。结果慢性弓形虫感染大鼠和正常对照组大鼠脊髓蛋白斑点数分别检出(268±13)个和(301±15)个,对2张电泳图进行匹配后发现有7个蛋白斑点在2组大鼠脊髓组织中的含量发生了3倍以上的变化,有差异的7个蛋白斑点经质谱鉴定的数据检索发现3个差异表达蛋白质,分别为甘油三磷酸脱氢酶(similar to glyceraldehyde-3-phosphate dehydrogenase)、含缬酪肽蛋白(valosin-containing protein)和类肌动蛋白[cytoplasmic 2(Gamma-actin)]。结论慢性弓形虫感染对大鼠脊髓神经的损伤机制可能与能量代谢受阻和细胞增殖有关。蛋白差异点有可能成为慢性弓形虫感染的治疗靶点。

Objectives To observe the proteome changes and differences in expression in the spinal cord of rats with a chronic Toxoplasma gondii infection and to explore the molecular basis of spinal cord injury due to chronic toxoplasmosis.Methods Six male SD rats were randomly divided into 2 groups,a control group and infection group.Each rat infected with T.gondii was intraperitoneally injected with 107/ml × 2 ml purified tachyzoites,and each rat in the control group was injected with 2 ml sterile saline.Rats were dissected after ten weeks.Total protein in the spinal cord was separated using two-dimensional gel electrophoresis.After Coomassie blue staining,the Image Master Image Analysis software was used to select and separate proteins on the gel.Matrix-assisted laser desorption ionization time-of-flight mass spectrometry（MALDI-TOF-MS） was used for peptide mass fingerprinting（PMF）.Proteins were identified by using Mascot software to search the MSDB and SWISS2PROT databases.Results Satisfactory 2-DE patterns were obtained for proteins in the spinal cord.In total,268±13 protein spots were obtained from the spinal cord of rats infected with T.gondii and 301±15 protein spots were obtained from the spinal cord of control rats.In 2-DE gels,7 spots increased or decreased in quantity.MALDL-TOF-MS identified 3 protein spots： a protein similar to glyceraldehyde-3-phosphate dehydrogenase,valosin-containing protein,and Gamma-actin.Conclusion The mechanisms of spinal cord injury due to chronic toxoplasmosis may relate to inhibition of energy metabolism and cell proliferation.Different protein spots may be a target for treatment of chronic toxoplasmosis.