用侵袭性曲霉病患者血清筛选特异性反应的烟曲霉蛋白质

Selection of specific immunoreactive proteins of Aspergillus fumigatus from sera of patients with invasive aspergillosis

目的用对烟曲霉蛋白质反应阳性的侵袭性曲霉病(IA)患者血清筛选免疫反应性最强的烟曲霉蛋白质用于免疫蛋白质组分析。方法 TCA/丙酮沉淀法制备烟曲霉分泌蛋白及菌体蛋白质作为包被抗原,用ELISA法筛选确诊IA患者血清,反应阳性的血清进一步与烟曲霉分泌蛋白及菌体蛋白质进行免疫印迹,筛选免疫反应最强的蛋白质。结果 11例确诊的IA患者中,6例血清抗体阳性。用此血清进行免疫印迹分析显示,烟曲霉分泌蛋白质的免疫反应性在反应强度及反应数量上均优于菌体蛋白质。在4种不同培养基的分泌蛋白中,YEPG分泌蛋白的免疫反应性最强,有免疫反应的蛋白条带数量最多。结论用YEPG培养烟曲霉14d的分泌蛋白质免疫反应性最强,且免疫反应条带最多,拟用于免疫蛋白质组学分析,进一步筛选特异性烟曲霉抗原。

Objective To screen the protein of Aspergillus fumigatus with the strongest immunoreactivity from the sera of invasive aspergillosis（IA） patients for immunoproteomic analysis.Methods The secreted proteins and mycelial protein of A.fumigatus prepared by TCA/acetone method were coated on ELISA plates.The antibody levels of A.fumigatus in the sera of diagnosed invasive aspergillosis（IA） patients were detected by ELISA.The secreted protein and mycelial protein of A.fumigatus were verified by immunoblot with the positive sera in ELISA.The strongest immunoreactive protein was screened.Results Among the 11 diagnosed IA patients 6 showed positive reaction against A.fumigatus antibody.The immunoblot results indicated that the immunoreactivity of secretory proteins was higher than that of mycelial proteins.Among 4 kinds of culture media,the proteins secreted in yeast peptone glucose medium（YEPG） showed the strongest reactive intensity and the maximum bands.Conclusions The protein secreted by A.fumigatus growing in YEPG for 14 days showed the strongest immunoreactivity and the most maximum bands,so the protein should be useful for further screening the specific antigens of A.fumigatus by immunoproteomic approach.