Exploring early and late Toxoplasma gondii strain RH infection by two-dimensional immunoblots of chicken immunoglobulin G and M profiles.
Exploring early and late Toxoplasma gondii strain RH infection by two-dimensional immunoblots of chicken immunoglobulin G and M profiles.
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DOI：
10.1371/journal.pone.0121647
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Suo X
中科院分区：
文献类型：
--
作者：
El-Ashram S;Sun X;Yin Q;Liu X;Suo X
弓形虫是一种细胞内顶复体寄生虫，感染温血脊椎动物宿主，只有早期感染阶段被药物控制。但无法区分早期和晚期感染的诊断。在本研究中，使用二维免疫印迹来探索鸡的早期和晚期感染。T.通过标准一次和常规双向凝胶聚丙烯酰胺电泳（2D-PAGE）分离的速殖子蛋白质组的图像分析来确定弓形虫。从T.刚地。速殖子蛋白质组的跨越pH 3-10范围的代表性凝胶由1306个不同的多肽斑点组成。应用双向电泳结合免疫印迹技术对弓形虫RH株免疫球蛋白（Ig）的M、G图谱进行解析和比较。速殖子总蛋白。用双向凝胶电泳分离弓形虫，并用Western印迹法分析它们与感染后7天和56天（dpi）SPF鸡抗血清的反应性。在用M和G免疫球蛋白分析期间检测到不同的抗原决定簇模式。在分析的多肽斑点总数（1306个差异表达的蛋白质斑点）中，6.97%被鉴定为在与IgG和IgM抗体的免疫印迹谱上具有共享的抗原多肽斑点，而不管感染后的时间。此外，一些免疫反应性多肽斑点似乎与感染的阶段有关。有趣的是，我们发现了天然抗体弓形虫抗原，除了高度保守的抗原决定簇，与非特异性二抗反应，山羊抗鸡IgG抗体与辣根过氧化物酶结合。总之，独特的反应性多肽斑点是有前途的候选人指定的分子标记，以区分早期和晚期鸡感染。
Toxoplasma gondii is an intracellular apicomplexan parasite infecting warm-blooded vertebrate hosts, with only early infection stage being contained with drugs. But diagnosis differencing early and late infection was not available. In the present investigation, 2-dimensional immunobloting was used to explore early and late infections in chickens. The protein expression of T. gondii was determined by image analysis of the tachyzoites proteome separated by standard-one and conventional two-dimentional gel polyacrylamide electrophoresis (2D- PAGE). Pooled gels were prepared from tachyzoites of T. gondii. A representative gel spanning a pH range of 3-10 of the tachyzoite proteome consisted of 1306 distinct polypeptide spots. Two-dimensional electrophoresis (2-DE) combined with 2-DE immunoblotting was used to resolve and compare immunoglobulins (Igs) M & G patterns against Toxoplasma gondii strain RH (mouse virulent strain). Total tachyzoite proteins of T. gondii were separated by two-dimensional gel electrophoresis and analyzed by Western blotting for their reactivity with the 7 and 56 days post-infection (dpi) SPF chicken antisera. Different antigenic determinant patterns were detected during analysis with M and G immunoglobulins. Of the total number of polypeptide spots analyzed (1306 differentially expressed protein spots), 6.97% were identified as having shared antigenic polypeptide spots on immunoblot profiles with IgG and IgM antibodies regardless the time after infection. Furthermore, some of the immunoreactive polypeptide spots seemed to be related to the stage of infection. Interestingly, we found natural antibodies to toxoplasmic antigens, in addition to the highly conserved antigenic determinants that reacted with non-specific secondary antibody; goat anti-chicken IgG antibodies conjugated with horseradish peroxidase. In conclusion, unique reactive polypeptide spots are promising candidates for designation of molecular markers to discriminate early and late chicken infection.
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