State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, People's Republic of China.
College of Animal Science and Technology, Tibet Agriculture & Animal Husbandry Univerity, Nyingchi, 860000, Tibet, People's Republic of China.
Vet Parasitol. 2020 Feb;278:109032. doi: 10.1016/j.vetpar.2020.109032. Epub 2020 Jan 17.
Eimeria tenella, belonging to protozoon, is the causative agent of cecal coccidiosis in chicken and causes enormous impacts for poultry industry. The surface antigens of apicomplexan parasites function as attachment and invasion in host-parasite interaction. Meanwhile, host immune response is triggered as a result of parasitic invasion. Immunogenicity and potency as a vaccinal candidate antigen of E. tenella surface antigen 4 (EtSAG4) have been unknown. Therefore, a gene segment of E. tenella EtSAG4 was amplified and transplanted to pET28a prokaryotic vector for recombinant protein expression. Similarly, pEGFP-N1 eukaryotic vectors with EtSAG4 gene segment (pEGFP-N1-EtSAG4) amplified in 293 T cells as DNA vaccines. Reverse transcription-polymerase chain reaction (RT-PCR) assay and western blot analysis were used to demonstrate successful expressions of EtSAG4 in Escherichia coli or 293 T cells. Subsequently, animal experiments (72 cobb broilers) were performed to evaluate immunoprotective between recombinant protein and DNA vaccine of E. tenella EtSAG4 using different immunizing doses (50 or 100 μg), respectively. Serum from chickens infected with E. tenella identified recombinant EtSAG4 (rEtSAG4) protein. Chickens vaccinated with either rEtSAG4 protein or pEGFP-N1-EtSAG4 plasmids both shown a significant increase in concentration of IFN-γ (p < 0.05) compared with control groups indicating production of cell-mediated immunity. Besides, pEGFP-N1-EtSAG4 plasmids motivated more intense immune responses for immunoglobulin Y (IgY) and interleukin 17 (IL-17) (p < 0.05) contrast to control groups. However, there was no increase in concentration of interleukin 10 (IL-10) and interleukin 4 (IL-4) for both rEtSAG4 protein and pEGFP-N1-EtSAG4 plasmids. Chickens vaccinated with rEtSAG4 protein or pEGFP-N1-EtSAG4 plasmids both show higher weight, lower oocyst output and mean lesion scores compared with infection control groups. The highest anticoccidial index (ACI) value of immunized groups was 168.24 from EGFP-N1-EtSAG4 plasmids (100 μg) group. Generally, EGFP-N1-EtSAG4 plasmids as DNA vaccines provided a more effective immunoprotective for chickens against E. tenalla than that of rEtSAG4 protein as subunit vaccines. EtSAG4 is a promising candidate antigen gene for development of coccidiosis vaccine.
柔嫩艾美耳球虫（Eimeria tenella）属于原生动物，是鸡盲肠球虫病的病原体，对家禽业造成了巨大的影响。顶复门寄生虫的表面抗原在宿主-寄生虫相互作用中作为附着和入侵的功能。同时，寄生虫的入侵会引发宿主的免疫反应。柔嫩艾美耳球虫表面抗原 4（EtSAG4）的免疫原性和作为疫苗候选抗原的效力尚不清楚。因此，扩增了柔嫩艾美耳球虫 EtSAG4 的基因片段，并将其移植到 pET28a 原核载体中进行重组蛋白表达。同样，扩增了 293T 细胞中的 EtSAG4 基因片段的 pEGFP-N1 真核载体（pEGFP-N1-EtSAG4）作为 DNA 疫苗。使用逆转录-聚合酶链反应（RT-PCR）检测和 Western blot 分析来证明 EtSAG4 在大肠杆菌或 293T 细胞中的成功表达。随后，使用不同的免疫剂量（50 或 100μg），在 72 只科宝肉鸡中进行了柔嫩艾美耳球虫 EtSAG4 的重组蛋白和 DNA 疫苗的免疫保护作用的动物实验。从感染柔嫩艾美耳球虫的鸡中鉴定出重组 EtSAG4（rEtSAG4）蛋白。与对照组相比，用 rEtSAG4 蛋白或 pEGFP-N1-EtSAG4 质粒接种的鸡均显著增加了干扰素-γ（IFN-γ）（p<0.05）的浓度，表明产生了细胞介导的免疫。此外，与对照组相比，pEGFP-N1-EtSAG4 质粒诱导了更强烈的免疫球蛋白 Y（IgY）和白细胞介素 17（IL-17）（p<0.05）的免疫反应。然而，rEtSAG4 蛋白和 pEGFP-N1-EtSAG4 质粒均未增加白细胞介素 10（IL-10）和白细胞介素 4（IL-4）的浓度。用 rEtSAG4 蛋白或 pEGFP-N1-EtSAG4 质粒接种的鸡的体重均高于感染对照组，卵囊排出量和平均病变评分均低于感染对照组。来自 pEGFP-N1-EtSAG4 质粒（100μg）组的免疫组的最高抗球虫指数（ACI）值为 168.24。一般来说，与 rEtSAG4 蛋白亚单位疫苗相比，pEGFP-N1-EtSAG4 质粒作为 DNA 疫苗为鸡提供了更有效的抗柔嫩艾美耳球虫免疫保护。EtSAG4 是一种很有前途的球虫病疫苗候选抗原基因。
