应用免疫蛋白质组学方法鉴定弓形虫病诊断抗原分子的研究

发布时间：2018-09-07 16:57

【摘要】：目的鉴定对弓形虫感染具有潜在诊断价值的弓形虫抗原分子。方法制备刚地弓形虫RH株速殖子全虫可溶性蛋白及弓形虫感染小鼠腹腔蛋白,经SDS-PAGE分离后采用半干电转移方法将其转移到硝酸纤维素膜上,再分别与22份弓形虫感染者血清进行Western blot,以22份健康人血清和其他寄生虫病患者血清作为对照,筛选能被弓形虫感染者血清特异性识别的蛋白组分;两种蛋白样本经二维电泳分离后采用Western blot筛选能被弓形虫感染者混合血清识别,但不能被健康人血清及其他寄生虫病患者血清识别的特异性斑点。切取SDS-PAGE胶上相应的特异性蛋白质斑点进行MALDI-TOF分析,鉴定蛋白斑点的基因。结果一维电泳及Western blot结果显示,弓形虫感染小鼠腹腔蛋白中的90、37、30、28和18ku组分分别可被部分弓形虫感染者血清识别,其中为28ku蛋白能被22份弓形虫感染病人血清中的15份所识别;速殖子全虫可溶性虫体蛋白中的90、70、67、56、48、39、37、30、28和18ku组分可被部分弓形虫感染者血清识别,其中28ku蛋白可被22份弓形虫感染者血清中的21份识别,22份健康人血清中有2份与28ku蛋白呈弱阳性反应,与其他寄生虫病患者血清无交叉反应。二维电泳Western blot阳性斑点的飞行质谱分析显示,弓形虫感染小鼠腹腔蛋白中的28ku组分为磷酸丙糖异构酶,弓形虫速殖子全虫可溶性蛋白中的28ku组分为GRA2和GRA7。结论弓形虫抗原与宿主抗体反应具有高度异质性,GRA2、GRA7及磷酸丙糖异构酶等分子的弓形虫感染者血清识别率高,此三种蛋白可作为建立具有高敏感性的弓形虫病特异性免疫诊断方法的抗原分子。
[Abstract]:Objective to identify the antigen molecules of Toxoplasma gondii with potential diagnostic value for Toxoplasma gondii infection. Methods the soluble protein of Toxoplasma gondii RH strain and the peritoneal protein of mice infected with Toxoplasma gondii were prepared and transferred to the nitrocellulose membrane by semi-dry electric transfer after SDS-PAGE isolation. Western blot, was performed on 22 sera of Toxoplasma gondii infected with Toxoplasma gondii and 22 sera from healthy people and other parasitic patients were used as controls to screen the protein components which could be specifically recognized by the sera of Toxoplasma gondii infected persons. Two kinds of protein samples were separated by two-dimensional electrophoresis and screened by Western blot. They could be identified by mixed serum of Toxoplasma gondii but not by specific spots in healthy sera and other parasitic patients. The specific protein spots on SDS-PAGE gel were digested for MALDI-TOF analysis to identify the gene of protein spots. Results the results of one-dimensional electrophoresis and Western blot showed that the components of 90 ~ (37) ~ (30) and 18ku of Toxoplasma gondii infected mice could be recognized by some sera of Toxoplasma gondii infected by Toxoplasma gondii, of which 28ku protein could be identified by 15 out of 22 sera of patients infected with Toxoplasma gondii (Toxoplasma gondii). In soluble body protein of T. tachyzoites, the components of 90, 70, 67, 56, 48, 39, 37, 30, 28, and 18ku, can be identified in sera of some infected individuals with Toxoplasma gondii (Toxoplasma gondii, Toxoplasma gondii). Of the 22 sera of Toxoplasma gondii infected with Toxoplasma gondii, 2 of 22 sera from healthy people showed weak positive reaction with 28ku protein, but no cross reaction with other parasitic patients. FMS analysis of Western blot positive spots in Toxoplasma gondii infected mice showed that the 28ku component in the peritoneal protein of mice infected with Toxoplasma gondii was divided into propanose phosphate isomerase, and the 28ku component in the soluble protein of Toxoplasma Tachyzoites was divided into GRA2 and GRA7.. Conclusion the sera of Toxoplasma gondii infected with Toxoplasma gondii antigen and host antibody are highly heterogeneous, such as GRA2GRA7 and Propranose phosphate isomerase, and the recognition rate of Toxoplasma gondii infected with Toxoplasma is high. These three proteins can be used as antigen molecules for the establishment of highly sensitive immunodiagnostic methods for Toxoplasma gondii (Toxoplasma gondii).
【作者单位】：卫生部寄生虫病预防与控制重点实验室江苏省寄生虫病分子生物学重点实验室江苏省血吸虫病防治研究所;中国疾病预防控制中心传染病预防控制所诊断室;
【基金】：国家重大科技专项(No.2012ZX10004220) 国家自然科学基金项目(No.81201316,30972581,30471515) 江苏省自然科学基金项目(No.BK2012544,BK2008110)
【分类号】：R531.8

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