用免疫蛋白质组学技术筛选烟曲霉分泌蛋白中的免疫优势抗原

Immunodominant antigen screening of secretory proteins from Aspergillus fumigatus by immunoproteomics

目的用免疫蛋白质组学技术筛选烟曲霉分泌蛋白中的免疫优势抗原,用于侵袭性曲霉病(IA)的诊断标志物及疗效监测。方法 YEPG培养基培养14 d的真菌分泌蛋白质进行二维电泳(2-DE)分离,用抗曲霉抗体阳性的IA确诊患者混合血清进行免疫印迹,再用质谱分析及生物信息学方法鉴定阳性反应点。结果 2-DE、免疫印迹结果显示,确诊IA患者血清与分泌蛋白质中多种抗原反应强烈。40个蛋白质点被抗体阳性IA患者血清识别。对40个蛋白质点质谱分析,成功鉴定了39个免疫反应阳性的蛋白质点,代表17种蛋白质,包括分泌型二肽基肽酶V(DppV)、NAD-依赖型苹果酸脱氢酶、硫氧还蛋白还原酶、醛脱氢酶、果糖二磷酸醛缩酶、3-羟丁酰辅酶A脱氢酶等。在鉴定出的17种蛋白质中,有2个为已知抗原,15个首次报道为免疫原性蛋白质。有7种免疫原性蛋白质显示有多个蛋白质点。且在新发现的免疫原性蛋白质中,2号蛋白质(共13个蛋白质点)免疫反应最强,鉴定结果为硫氧还蛋白还原酶。结论免疫蛋白质组学技术是筛选病原体免疫优势抗原的一种有效方法,此次鉴定的抗原可能是潜在的疾病标志物,但还需进一步地实验加以评估。

Objective To screen the immunodominant antigens of secretory proteins from Aspergillus fumigatus by immunoproteomic in order to serve as the marker of clinical diagnosis and effectiveness of therapy for invasive aspergillosis.Methods The proteins secreted by Aspergillus fumigatus which grew in YEPG media for 14 days were separated by two-dimensional PAGE（2-DE）,and then transferred to PVDF membranes.Immunoblot was performed by using the mixed sera from definitely diagnostic patients with invasive aspergillosis.The immunoreactive spots were identified by mass spectrometric analysis and bioinformatics method.Results 2-DE immunobloting showed that the sera from invasive aspergillosis patients reacted strongly with a series of antigens in the secretory proteins.Forty dots were recognized by sera of patients and were analyzed by mass spectrometry.Among them,39 immunoreactive spots were successfully identified,which were belonged to 17 corresponding proteins,including dipeptidyl peptidase,NAD-dependent malate dehydrogenase,thioredoxin reductase,aldehyde dehydrogenase,fructose-bisphosphate aldolase,3-hydroxybutyryl-CoA dehydrogenase,etc.In the 17 proteins,2 were reported before and 15 were newly reportedimmunogenetic antigens.Seven immunogenic proteins displayed multiple spots.The novel immunogenic protein No.2,which contained 13 spots,showed the highest immunoreactivity and was identified as thioredoxin reductase.Conclusion The immunoproteomic technology should be effective approach for screening immunodominant antigens of pathogens.The antigens identified in this study might serve as the potential markers for the infection of Aspergillus fumigatus,but the diagnostic values have to be further evaluated.