College of Veterinary Medicine, Northeast Agricultural University, NO. 600 Changjiang Road, Xiangfang District, Harbin, Heilongjiang, 150030, China.
Heilongjiang Key Laboratory for Experimental Animals and Comparative Medicine, Harbin, Heilongjiang, 150030, China.
Vet Res. 2021 Feb 17;52(1):24. doi: 10.1186/s13567-020-00873-y.
Avian coccidiosis caused by Eimeria leads to huge economic losses on the global poultry industry. In this study, microneme adhesive repeat regions (MARR) bc1 of E. tenella microneme protein 3 (EtMIC3-bc1) was used as ligand, and peptides binding to EtMIC3 were screened from a phage display peptide library. The positive phage clones were checked by enzyme-linked immunosorbent assay (ELISA). Competitive ELISA was applied to further verify the binding capability between the positive phages and recombinant EtMIC3-bc1 protein or sporozoites protein. The inhibitory effects of target peptides on sporozoites invasion of MDBK cells were measured in vitro. Chickens were orally administrated with target positive phages and the protective effects against homologous challenge were evaluated. The model of three-dimensional (3D) structure for EtMIC3-bc1 was conducted, and molecular docking between target peptides and EtMIC3-bc1 model was analyzed. The results demonstrated that three selected positive phages specifically bind to EtMIC3-bc1 protein. The three peptides A, D and W effectively inhibited invasion of MDBK cells by sporozoites, showing inhibited ratio of 71.8%, 54.6% and 20.8%, respectively. Chickens in the group orally inoculated with phages A displayed more protective efficacies against homologous challenge than other groups. Molecular docking showed that amino acids in three peptides, especially in peptide A, insert into the hydrophobic groove of EtMIC3-bc1 protein, and bind to EtMIC3-bc1 through intermolecular hydrogen bonds. Taken together, the results suggest EtMIC3-binding peptides inhibit sporozoites entry into host cells. This study provides new idea for exploring novel strategies against coccidiosis.
鸡球虫病由艾美耳属球虫引起，给全球家禽业造成了巨大的经济损失。本研究以柔嫩艾美耳球虫微线蛋白 3（EtMIC3）的 MARRbc1 区作为配体，从噬菌体展示肽库中筛选与 EtMIC3 结合的肽段。通过酶联免疫吸附试验（ELISA）检测阳性噬菌体克隆。竞争 ELISA 进一步验证了阳性噬菌体与重组 EtMIC3-bc1 蛋白或子孢子蛋白的结合能力。体外测定目标肽段对 MDBK 细胞子孢子入侵的抑制作用。鸡经口给予目标阳性噬菌体，评估其对同源性挑战的保护作用。构建 EtMIC3-bc1 三维（3D）结构模型，分析目标肽段与 EtMIC3-bc1 模型的分子对接。结果表明，3 个选定的阳性噬菌体特异性结合 EtMIC3-bc1 蛋白。3 个肽段 A、D 和 W 有效抑制子孢子入侵 MDBK 细胞，抑制率分别为 71.8%、54.6%和 20.8%。口服噬菌体 A 的鸡对同源性挑战的保护效果优于其他组。分子对接表明，3 个肽段中的氨基酸，特别是肽段 A，插入到 EtMIC3-bc1 蛋白的疏水槽中，通过分子间氢键与 EtMIC3-bc1 结合。总之，结果表明 EtMIC3 结合肽抑制子孢子进入宿主细胞。本研究为探索抗球虫病的新策略提供了新的思路。
