柔嫩艾美耳球虫顶膜抗原-1潜在子孢子互作蛋白的筛选

发布时间：2018-06-28 05:53

本文选题：柔嫩艾美耳球虫 + 顶膜抗原-　；参考：《中国动物传染病学报》2017年03期

【摘要】：为了筛选与柔嫩艾美耳球虫顶膜抗原-1(Eimeria tenella apical membrance antigen 1,EtAMA1)存在互作的子孢子蛋白,以柔嫩艾美耳球虫子孢子c DNA为模板,PCR扩增EtAMA1基因胞外区,将扩增产物与p GEX-6P-1连接,构建原核表达质粒p GEX-6P-1-EtAMA1,表达、纯化重组蛋白EtAMA1-GST。纯化的重组蛋白经Western blot验证,结果显示在75 k Da处有单一的目的条带,证实了所获得的蛋白为EtAMA1-GST融合蛋白。将EtAMA1-GST蛋白、GST蛋白和PBS分别与谷胱甘肽琼脂糖珠孵育后,再分别与子孢子裂解物共孵育,洗脱液经SDS-PAGE分析后,结果显示三者之间的条带明显不同。将差异条带切下进行质谱分析,对获得的蛋白质肽段与Uniprot中鸡球虫蛋白质数据库进行Blast比对,初步获得了10个与EtAMA1存在潜在互作的柔嫩艾美耳球虫子孢子蛋白,包括微线体蛋白、糖酵解酶类、肌动蛋白相关因子以及一些保守蛋白等,他们广泛参与了虫体的入侵、发育、能量代谢等生物学过程。研究结果为进一步阐明EtAMA1在球虫入侵宿主细胞中发挥重要作用的分子机制奠定了基础。
[Abstract]:In order to screen the sporozoite proteins interacting with Eimeria tenella apical membrance antigen 1 and EtAMA1, the extracellular region of EtAMA1 gene was amplified by PCR using Eimeria tenella apical membrance antigen 1 and EtAMA1 as template, and the product was linked to pGEX-6P-1. The prokaryotic expression plasmid pGEX-6P-1-EtAMA1 was constructed, and the recombinant protein EtAMA1-GST-1 was expressed and purified. The purified recombinant protein was confirmed as EtAMA1-GST fusion protein by Western blot. The result showed that there was a single target band at 75 kDa. The GST and PBS of EtAMA1-GST protein were incubated with glutathione agarose beads, and then incubated with the sporozoite lysates respectively. The eluent was analyzed by SDS-PAGE. The results showed that the bands of the three proteins were obviously different. The differential bands were cut off and analyzed by mass spectrometry. The obtained protein peptides were compared with the chicken coccidia protein database in Uniprot by blast. Ten Eimeria tenella spores proteins with potential interaction with EtAMA1 were obtained. Including microsomal proteins, glycolytic enzymes, actin related factors and some conserved proteins, they are widely involved in the biological processes of insect invasion, development, energy metabolism and so on. The results provide a basis for further elucidation of the molecular mechanism of EtAMA1 which plays an important role in the invasion of host cells by coccidia.
【作者单位】：中国农业科学院上海兽医研究所农业部动物寄生虫学重点开放实验室;江苏省动物重要疫病与人兽共患病防控协同创新中心;上海师范大学生命与环境科学学院;
【基金】：国家自然科学基金(31672551,31272557) 上海市闵行区人才发展专项基金(201351)
【分类号】：S852.723

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