柔嫩艾美耳球虫顶膜抗原-1潜在子孢子互作蛋白的筛选

SCREENING OF THE POTENTIAL SPOROZOITE PROTEINS INTERACTING WITH EIMERIA TENELLA APICAL MEMBRANCE ANTIGEN-1

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。

In order to screen the potential sporozoite proteins interacting with Eimeria tenella apical membrance antigen 1 （EtAMA 1）, the ectodomain of EtAMAlwas amplified in PCR with the cDNAs of the E. tenella sporozoite as template. The PCR products were subcloned to pGEX-6P-1 vector for expression of the recombinant protein. The purified recombinant protein EtAMA 1-GST was identified in Western blot. The results showed that a protein about 75 kDa in accordance with molecular weight of target protein was recognized by anti-GST antibody, which confirmed that the expressed product was EtAMA1-GST fusion protein. Glutathione sepharose beads were incubated with EtAMA1-GST, GST protein or PBS and then the beads were incubated with sporozoite lysates. Bound proteins were eluted by using the sample buffer and eluants were subject to SDS-PAGE. The separated bands were digested and analyzed by mass spectrometry. The resulting protein peptides were blasted with Eimeria proteins database deposited in Uniprot and 10 potential sporozoite proteins were obtained. These proteins, mainly including microneme proteins, glycolytic enzymes, actin-related proteins and some hypothetical proteins, are involved in many molecular processes, such as invasion, development and energy metabolism. These results were informative for further elucidating the molecular mechanism of EtAMA1 that plays an important role inEimeria host invasion.