Zhu Yu, Liu Dandan, Wang Lele, Feng Qianqian, Xue Nianyu, Hou Zhaofeng, Xu Jinjun, Tao Jianping
College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China.
Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China.
Animals (Basel). 2025 Aug 23;15(17):2482. doi: 10.3390/ani15172482.
SSB proteins play essential roles in DNA replication, recombination, and repair in bacteria, archaea, and eukarya. This study investigates the transcript levels, identification, expression and purification, subcellular localization, and immune protective potential of the SSB-like proteins of (EnSSB), exploring its role in the development of and its potential as a candidate antigen for a subunit vaccine against avian coccidiosis. The level of EnSSB gene transcription was highest in unsporulated oocysts (UO), followed by gametocytes (GAM) ( < 0.05). The gene consisted of an open reading frame of 1488 nucleotides encoding a protein of 495 amino acid residues with a predicted molecular weight of 53.31 kDa. EnSSB contained a SSB domain with a conserved OB (oligonucleotide/oligosaccharide binding) fold. The molecular mass of the native protein, as determined by Western blot analysis, was 58 kDa in second-generation merozoites (MZ-2) and UO. In addition to the 58 kDa band, four other bands (98 kDa, ~82 kDa, ~36 kDa and ~28 kDa) were detected in GAM. No bands were detected in MZ-3. Indirect immunofluorescence and immuno-electron microscopy localized EnSSB in the cytoplasm of macrogametocytes but not in wall-forming bodies and oocyst wall. Animal challenge experiments demonstrated that rEnSSB elicited robust IgY responses, increased splenic T lymphocytes and body weight gain, reduced intestinal lesion scores and oocyst shedding, and presented anticoccidial index (ACI) more than 160. These findings not only offer a foundation for understanding the role of EnSSB protein in regulating the development of , but also present a potential protective antigen of for the development of a subunit vaccine against avian coccidiosis.
单链DNA结合蛋白（SSB蛋白）在细菌、古细菌和真核生物的DNA复制、重组及修复过程中发挥着至关重要的作用。本研究对鸡球虫（EnSSB）的类SSB蛋白的转录水平、鉴定、表达与纯化、亚细胞定位以及免疫保护潜力进行了探究，以明确其在鸡球虫发育过程中的作用以及作为抗鸡球虫亚单位疫苗候选抗原的潜力。EnSSB基因转录水平在未孢子化卵囊（UO）中最高，其次是配子体（GAM）（P < 0.05）。该基因由一个1488个核苷酸的开放阅读框组成，编码一个495个氨基酸残基的蛋白质，预测分子量为53.31 kDa。EnSSB包含一个具有保守寡核苷酸/寡糖结合（OB）折叠的SSB结构域。通过蛋白质免疫印迹分析测定，天然蛋白在第二代裂殖子（MZ - 2）和UO中的分子量约为58 kDa。除了58 kDa的条带外，在GAM中还检测到另外四条条带（约98 kDa、约82 kDa、约36 kDa和约28 kDa）。在MZ - 3中未检测到条带。间接免疫荧光和免疫电子显微镜观察表明，EnSSB定位于大配子体的细胞质中，而非壁状体和卵囊壁中。动物攻毒实验表明，重组EnSSB能引发强烈的IgY反应，增加脾脏T淋巴细胞数量和体重增加，降低肠道病变评分和卵囊排出量，抗球虫指数（ACI）超过160。这些发现不仅为理解EnSSB蛋白在调控鸡球虫发育中的作用提供了基础，也为开发抗鸡球虫亚单位疫苗提供了一种潜在的保护性抗原。
