Juárez-Estrada Marco A, Tellez-Isaias Guillermo, Graham Danielle M, Laverty Lauren, Gayosso-Vázquez Amanda, Alonso-Morales Rogelio A
Departamento de Medicina y Zootecnia de Aves, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Mexico City, Mexico.
Departamento de Genética y Bioestadística, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Mexico City, Mexico.
Front Vet Sci. 2023 Jul 24;10:1223436. doi: 10.3389/fvets.2023.1223436. eCollection 2023.
Coccidiosis, caused by parasites of numerous species, has long been recognized as an economically significant disease in the chicken industry worldwide. The rise of anti-coccidian resistance has driven a search for other parasite management techniques. Recombinant antigen vaccination presents a highly feasible alternative. Properly identifying antigens that might trigger a potent immune response is one of the major obstacles to creating a viable genetically modified vaccine.
This study evaluated a reverse immunology approach for the identification of B-cell epitopes. Antisera from rabbits and hens inoculated with whole-sporozoites of were used to identify Western blot antigens. The rabbit IgG fraction from the anti-sporozoite serum exhibited the highest reactogenicity; consequently, it was purified and utilized to screen two random Phage-display peptide libraries (12 mer and c7c mer). After three panning rounds, 20 clones from each library were randomly selected, their nucleotide sequences acquired, and their reactivity to anti-sporozoite serum assessed. The selected peptide clones inferred amino acid sequences matched numerous proteins.
The extracellular domain of the epidermal growth factor-like (EGF-like) repeats, and the thrombospondin type-I (TSP-1) repeats of micronemal protein 4 (EtMIC4) matched with the c7c mer selected clones CNTGSPYEC (2/20) and CMSTGLSSC (1/20) respectively. The clone CSISSLTHC that matched with a conserved hypothetical protein of was widely selected (3/20). Selected clones from the 12-mer phage display library AGHTTQFNSKTT (7/20), GPNSAFWAGSER (2/20) and HFAYWWNGVRGP (8/20) showed similarities with a cullin homolog, elongation factor-2 and beta-dynein chain a putative protein, respectively. Four immunodominant clones were previously selected and used to immunize rabbits. By ELISA and Western blot, all rabbit anti-clone serums detected native antigens.
Thus, selected phagotopes contained recombinant antigen peptides. Using antibodies against sporozoites, this study demonstrated the feasibility of screening Phage-display random peptide libraries for true immunotopes. In addition, this study looked at an approach for finding novel candidates that could be used as an recombinant epitope-based vaccine.
球虫病由多种寄生虫引起，长期以来一直被认为是全球养鸡业中具有重要经济意义的疾病。抗球虫药耐药性的增加促使人们寻找其他寄生虫管理技术。重组抗原疫苗是一种非常可行的替代方法。正确识别可能引发有效免疫反应的抗原是开发可行的转基因疫苗的主要障碍之一。
本研究评估了一种用于鉴定B细胞表位的反向免疫学方法。用接种了 全子孢子的兔和母鸡的抗血清来鉴定蛋白质印迹抗原。抗子孢子血清中的兔IgG组分表现出最高的反应原性；因此，将其纯化并用于筛选两个随机噬菌体展示肽库（12肽库和c7c肽库）。经过三轮淘选后，从每个库中随机选择20个克隆，获取其核苷酸序列，并评估它们与抗子孢子血清的反应性。所选肽克隆推断的氨基酸序列与多种 蛋白质匹配。
表皮生长因子样（EGF样）重复序列的胞外结构域以及微小膜蛋白4（EtMIC4）的血小板反应蛋白I型（TSP-1）重复序列分别与c7c肽库中选择的克隆CNTGSPYEC（2/20）和CMSTGLSSC（1/20）匹配。与 的一个保守假设蛋白匹配的克隆CSISSLTHC被广泛选择（3/20）。从12肽噬菌体展示库中选择的克隆AGHTTQFNSKTT（7/20）、GPNSAFWAGSER（2/20）和HFAYWWNGVRGP（8/20）分别与一种cullin同源物、延伸因子-2和β-动力蛋白链一种假定 蛋白具有相似性。先前选择了四个免疫显性克隆并用于免疫兔子。通过ELISA和蛋白质印迹，所有兔抗克隆血清均检测到 天然抗原。
因此,所选噬菌体位点包含重组 抗原肽。本研究利用抗 子孢子抗体，证明了筛选噬菌体展示随机肽库以寻找真正免疫表位的可行性。此外,本研究探讨了一种寻找可作为基于重组表位疫苗的新候选物的方法。
