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.
Biology Department, Yunnan University, Kunming, 650500, China.
Mol Biochem Parasitol. 2020 Nov;240:111318. doi: 10.1016/j.molbiopara.2020.111318. Epub 2020 Sep 18.
Gametocyte proteins of Eimeria spp. are essential components of the oocyst wall, and some of these proteins have been analysed to identify targets of transmission-blocking vaccines against avian coccidiosis. In the present study, a cDNA from E. necatrix gametocytes was cloned and sequenced. The cDNA is 1473 bp in length and encodes a 490-amino-acid protein containing a tyrosine-serine (Tyr/Ser)-rich domain and a proline-methionine (Pro/Met)-rich domain. A quantitative real-time PCR (qPCR) analysis showed that the cDNA is expressed only during gametogenesis. A fragment containing the Tyr/Ser-rich domain (rEnGAM59) was expressed in Escherichia coli BL21 (DE3) cells. Immunoblotting showed that rEnGAM59 was recognized by the serum of convalescent chickens after infection with E. necatrix, and that an anti-rEnGAM59 antibody recognized a ∼59 kDa protein and two other proteins (∼35 kDa and ∼33 kDa) in gametocyte extracts. An immunofluorescence assay showed that the anti-rEnGAM59 antibody recognized wall-forming bodies in the macrogametocytes and oocyst walls. An in vivo vaccination and challenge trial was conducted to test the potential utility of rEnGAM59 as a vaccine. Immunized chickens performed better than the unimmunized and challenged (positive control) chickens. The intestinal lesion scores were significantly lower in the immunized groups than in the positive control group (P < 0.05). In contrast, the body weight gains (BWG) were significantly higher in the immunized groups than in the positive control group (P < 0.05). There were no significant differences in the lesion scores and BWG between the groups immunized with rEnGAM59 protein or with live oocysts (P> 0.05). Chickens immunized with rEnGAM59 protein had a significantly higher antigen-specific serum IgY response (P < 0.05). rEnGAM59 protein can be used as candidate antigen to develop a recombinant coccidiosis vaccine.
柔嫩艾美耳球虫配子体蛋白是卵囊壁的重要组成部分，其中一些蛋白已被分析用于鉴定抗禽球虫病的传播阻断疫苗的靶标。本研究克隆并测序了柔嫩艾美耳球虫配子体的 cDNA。该 cDNA 长 1473bp，编码一个 490 个氨基酸的蛋白，含有一个酪氨酸-丝氨酸（Tyr/Ser）丰富域和一个脯氨酸-蛋氨酸（Pro/Met）丰富域。定量实时 PCR（qPCR）分析表明，该 cDNA 仅在配子发生过程中表达。含有 Tyr/Ser 丰富域的片段（rEnGAM59）在大肠杆菌 BL21（DE3）细胞中表达。免疫印迹显示，rEnGAM59 被感染柔嫩艾美耳球虫后的康复鸡血清识别，抗 rEnGAM59 抗体识别配子体提取物中的一个约 59 kDa 蛋白和另外两个蛋白（约 35 kDa 和约 33 kDa）。免疫荧光分析显示，抗 rEnGAM59 抗体识别大配子体和卵囊壁中的壁形成体。进行了体内免疫接种和攻毒试验以测试 rEnGAM59 作为疫苗的潜在用途。免疫鸡的表现优于未免疫和攻毒（阳性对照）的鸡。免疫组的肠道病变评分明显低于阳性对照组（P<0.05）。相比之下，免疫组的体重增加（BWG）明显高于阳性对照组（P<0.05）。rEnGAM59 蛋白免疫组与活卵囊免疫组之间的病变评分和 BWG 无显著差异（P>0.05）。rEnGAM59 蛋白免疫鸡的抗原特异性血清 IgY 反应明显更高（P<0.05）。rEnGAM59 蛋白可用作开发重组球虫病疫苗的候选抗原。
