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; Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China.
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; Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China.
Vet Parasitol. 2023 Sep;321:110002. doi: 10.1016/j.vetpar.2023.110002. Epub 2023 Aug 1.
Eimeria species are intracellular obligate parasites, among the most common pathogens affecting the intensive poultry industry. Oxidoreductases are members of a class of proteins with redox activity and are widely found in apicomplexan protozoans. However, there have been few reports related to Eimeria species. In this study, total RNA was extracted from the gametocytes of E. necatrix Yangzhou strain to amplify the EnOXIO1 gene using reverse-transcription polymerase chain reaction. After cloning and sequence analysis, the prokaryotic expression vector pET-28a(+)-EnOXIO1 was constructed and transformed into Escherichia coli BL21(DE3), and the recombinant protein rEnOXIO1 was expressed by induction with isopropyl ß-D-1-thiogalactopyranoside. The full length EnOXIO1 gene was 2535 bp encoding 844 amino acids, and the EnOXIO1 protein had a molecular weight of about 100 kDa and was mainly expressed in inclusion bodies. Western blot analysis indicated that the rEnOXIO1 protein had good antigenicity and cross-reactivity and was specifically recognized by a 6 ×HIS labeled monoclonal antibody, mouse anti-recombinant protein polyclonal antibody, and recovery serum from chickens infected with E. necatrix, E. acervulina, and E. tenella sporulated oocysts. The results of laser confocal immunofluorescence localization showed that the EnOXIO1 protein was mainly located on the wall-forming bodies in gametocytes and played an important role in the formation of the oocyst wall. Quantitative PCR analysis revealed that transcript levels of EnOXIO1 were highest in the gametocyte stage. Protein expression levels of EnOXIO1 were higher in the gametocyte stage than in other developmental stages according to western blot analysis. Vaccination of chickens against E. necatrix was achieved with recombinant protein rEnOXIO1, which triggered humoral immunity and antibody production, increased average body weight gain, reduced oocyst output and alleviated lesions after E. necatrix infection. The highest ACI value (172.36) was observed in chickens that received 200 μg rEnOXIO1 compared with other immunization groups.
艾美耳球虫是一种细胞内专性寄生虫，是影响集约化家禽养殖业的最常见病原体之一。氧化还原酶是具有氧化还原活性的蛋白质中的一类成员，广泛存在于顶复门原生动物中。然而，关于艾美耳球虫的报道很少。本研究从扬州株柔嫩艾美耳球虫的配子体中提取总 RNA，采用反转录聚合酶链反应扩增 EnOXIO1 基因。克隆和序列分析后，构建了原核表达载体 pET-28a(+)-EnOXIO1，并转化至大肠杆菌 BL21(DE3)中，异丙基 β-D-1-硫代半乳糖苷诱导表达重组蛋白 rEnOXIO1。全长 EnOXIO1 基因长 2535bp，编码 844 个氨基酸，EnOXIO1 蛋白分子量约为 100kDa，主要以包涵体形式表达。Western blot 分析表明，rEnOXIO1 蛋白具有良好的抗原性和交叉反应性，可被 6×HIS 标记的单克隆抗体、鼠抗重组蛋白多克隆抗体以及感染柔嫩艾美耳球虫、堆形艾美耳球虫和毒害艾美耳球虫孢子化卵囊的鸡血清特异性识别。激光共聚焦免疫荧光定位结果显示，EnOXIO1 蛋白主要位于配子体的壁形成体上，在卵囊壁的形成中发挥重要作用。定量 PCR 分析显示，EnOXIO1 的转录水平在配子体阶段最高。Western blot 分析显示，与其他发育阶段相比，EnOXIO1 蛋白在配子体阶段的表达水平更高。用重组蛋白 rEnOXIO1 对鸡进行免疫接种，可诱导体液免疫和抗体产生，增加平均体重增加，减少卵囊排出量，并减轻感染柔嫩艾美耳球虫后的病变。与其他免疫组相比，接受 200μg rEnOXIO1 免疫的鸡的 ACI 值（172.36）最高。
