Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, CAAS, 200241 Shanghai, PR China.
Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, CAAS, 200241 Shanghai, PR China - College of Life and Environment Sciences, Shanghai Normal University, 200234 Shanghai, PR China.
Parasite. 2021;28:40. doi: 10.1051/parasite/2021037. Epub 2021 May 3.
Eimeria tenella is an obligate intracellular parasite that actively invades cecal epithelial cells of chickens. This parasite encodes a genome of more than 8000 genes. However, more than 70% of the gene models for this species are currently annotated as hypothetical proteins. In this study, a conserved hypothetical protein gene of E. tenella, designated EtCHP18905, was cloned and identified, and its immune protective effects were evaluated. The open reading frame of EtCHP18905 was 1053bp and encoded a protein of 350 amino acids with a molecular weight of 38.7kDa. The recombinant EtCHP18905 protein (rEtCHP18905) was expressed in E. coli. Using western blot, the recombinant protein was successfully recognized by anti GST-Tag monoclonal antibody and anti-sporozoites protein rabbit serum. Real-time quantitative PCR analysis revealed that the EtCHP18905 mRNA levels were higher in sporozoites than in unsporulated oocysts, sporulated oocysts and second-generation merozoites. Western blot analysis showed that EtCHP18905 protein expression levels were lower in sporozoites than in other stages. Immunofluorescence analysis indicated that the EtCHP18905 protein was located on the surface of sporozoites and second-generation merozoites. Inhibition experiments showed that the ability of sporozoites to invade host cells was significantly decreased after treatment with the anti-rEtCHP18905 polyclonal antibody. Vaccination with rEtCHP18905 protein was able to significantly decrease mean lesion scores and oocyst outputs as compared to non-vaccinated controls. The results suggest that the rEtCHP18905 protein can induce partial immune protection against infection with E. tenella and could be an effective candidate for the development of new vaccines.
柔嫩艾美耳球虫是一种专性细胞内寄生虫，它能主动侵袭鸡的盲肠上皮细胞。该寄生虫的基因组超过 8000 个基因。然而，该物种超过 70%的基因模型目前被注释为假设蛋白。在这项研究中，克隆并鉴定了柔嫩艾美耳球虫的一个保守的假设蛋白基因，命名为 EtCHP18905，并评估了其免疫保护作用。EtCHP18905 的开放阅读框为 1053bp，编码一个 350 个氨基酸的蛋白质，分子量为 38.7kDa。重组 EtCHP18905 蛋白（rEtCHP18905）在大肠杆菌中表达。通过 Western blot，重组蛋白被抗 GST-Tag 单克隆抗体和抗裂殖体蛋白兔血清成功识别。实时定量 PCR 分析显示，EtCHP18905mRNA 水平在子孢子中高于未孢子化卵囊、孢子化卵囊和第二代裂殖体。Western blot 分析显示，EtCHP18905 蛋白表达水平在子孢子中低于其他阶段。免疫荧光分析表明，EtCHP18905 蛋白位于子孢子和第二代裂殖体的表面。抑制实验表明，用抗 rEtCHP18905 多克隆抗体处理后，子孢子侵入宿主细胞的能力显著降低。与未接种对照组相比，rEtCHP18905 蛋白疫苗接种能显著降低平均病变评分和卵囊产量。结果表明，rEtCHP18905 蛋白能诱导对柔嫩艾美耳球虫感染的部分免疫保护，可能是开发新型疫苗的有效候选物。
