柔嫩艾美耳球虫棒状体颈部蛋白Et RON2 L1-2特性与功能初步分析

Characteristics and Biological Function of Rhoptry Neck Protein RON2 L1-2 of Eimeria tenella

为研究柔嫩艾美耳球虫棒状体颈部蛋白2(RON2)同源分子——Et RON2 L1-2的分子特性与功能,利用实时荧光定量PCR、Western blot、入侵抑制试验、间接免疫荧光试验以及双分子荧光互补实验(BiFC)等方法分析其生物学特征。结果发现Et RON2 L1-2蛋白序列与Et RON2仅有29.69%的同源性,转录水平和蛋白水平均在第二代裂殖子阶段最高。抗r Et RON2 L1-2抗体对子孢子入侵细胞有明显抑制作用。Et RON2 L1-2蛋白主要分布于经PBS孵育子孢子的胞质和表面,并分布于经完全培养基孵育或入侵DF-1细胞后2 h子孢子的顶端;其表达量在滋养体和未成熟裂殖体增加,且在带虫空泡膜也有分布;在第二代裂殖子中主要分布于整个胞质。Et RON2 L1-2与柔嫩艾美耳球虫顶膜抗原4(AMA4)之间存在互作关系。

In order to investigate the molecular characteristics and functions of Et RON2 L1-2,a Rhoptry neck protein 2 homologous molecule of Eimeria tenella,its biological characteristics were analyzed by quantitative real-time PCR,western blot,invasion inhibition assay,indirect immunofluorescence assay,and BiFC.The results showed that Et RON2 L1-2 protein sequence was only 29.69%identical to Et RON2,and the mRNA level and protein level were the highest at the second-generation merozoite stage.Anti-r Et RON2 L1-2 antibody significantly inhibited sporozoite invasion cells.The Et RON2 L1-2 protein was located at both the cytoplasm and the surface of the sporozoites incubated in PBS,and was mainly located in the apical part of sporozoites when incubated in complete medium or invaded DF-1 cells for 2 h;its expression was increased in the trophozoite and immature merozonts,and was distributed in the parasitophorous vacuolar membranes and in the whole cytoplasm of second-generation merozoite.There was an interaction between Et RON2 L1-2 and E.tenella apical membrane antigen 4(Et AMA4).