Yeng Chen, Osman Emelia, Mohamed Zeehaida, Noordin Rahmah
Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia.
Electrophoresis. 2010 Dec;31(23-24):3843-9. doi: 10.1002/elps.201000038.
Toxoplasma gondii infection in pregnant women may result in abortion and foetal abnormalities, and may be life-threatening in immunocompromised hosts. To identify the potential infection markers of this disease, 2-DE and Western blot methods were employed to study the parasite circulating antigens and host-specific proteins in the sera of T. gondii-infected individuals. The comparisons were made between serum protein profiles of infected (n=31) and normal (n=10) subjects. Antigenic proteins were identified by immunoblotting using pooled sera and monoclonal anti-human IgM-HRP. Selected protein spots were characterised using mass spectrometry. Prominent differences were observed when serum samples of T. gondii-infected individuals and normal controls were compared. A significant up-regulation of host-specific proteins, α(2)-HS glycoprotein and α(1)-B glycoprotein, was also observed in the silver-stained gels of both active and chronic infections. However, only α(2)-HS glycoprotein and α(1)-B glycoprotein in the active infection showed immunoreactivity in Western blots. In addition, three spots of T. gondii proteins were detected, namely (i) hypothetical protein chrXII: 3984434-3 TGME 49, (ii) dual specificity protein phosphatase, catalytic domain TGME 49 and (iii) NADPH-cytochrome p450 reductase TGME 49. Thus, 2-DE approach followed by Western blotting has enabled the identification of five potential infection markers for the diagnosis of toxoplasmosis: three are parasite-specific proteins and two are host-specific proteins.
孕妇感染弓形虫可能导致流产和胎儿畸形,在免疫功能低下的宿主中可能危及生命。为了确定该疾病的潜在感染标志物,采用二维电泳(2-DE)和蛋白质印迹法研究弓形虫感染个体血清中的寄生虫循环抗原和宿主特异性蛋白。对感染组(n = 31)和正常组(n = 10)受试者的血清蛋白谱进行了比较。通过使用混合血清和单克隆抗人IgM-HRP进行免疫印迹来鉴定抗原蛋白。使用质谱对选定的蛋白斑点进行表征。比较弓形虫感染个体和正常对照的血清样本时观察到显著差异。在急性和慢性感染的银染凝胶中还观察到宿主特异性蛋白α(2)-HS糖蛋白和α(1)-B糖蛋白显著上调。然而,在蛋白质印迹中只有急性感染中的α(2)-HS糖蛋白和α(1)-B糖蛋白显示出免疫反应性。此外,检测到三个弓形虫蛋白斑点,即(i)假设蛋白chrXII: 3984434-3 TGME 49,(ii)双特异性蛋白磷酸酶催化结构域TGME 49,(iii)NADPH-细胞色素P450还原酶TGME 49。因此,二维电泳方法结合蛋白质印迹能够鉴定出五种潜在的弓形虫病诊断感染标志物:三种是寄生虫特异性蛋白,两种是宿主特异性蛋白。