鸡球虫共同抗原UCE免疫原性分析及其对多种球虫感染的免疫保护效果评估

[目的]在前期研究中，筛选到了存在于多种鸡球虫间的共同抗原泛素缀合酶结构域蛋白（UCE），本研究进一步分析其免疫原性，评估其对多种球虫感染的免疫保护效果。[方法]提取纯化的堆型艾美耳球虫孢子化卵囊总RNA，用反转录PCR（RT-PCR）技术扩增堆型艾美耳球虫UCE基因（EaUCE），插入pVAX1质粒，构建真核表达质粒pVAX-EaUCE，通过RT-PCR和Western blot检测pVAX-EaUCE在鸡体内的转录和表达情况。经流式细胞术和间接ELISA法检测pVAX-EaUCE免疫后鸡体细胞免疫和体液免疫水平变化，分析其免疫原性。分别采用柔嫩艾美耳球虫、堆型艾美耳球虫、巨型艾美耳球虫及三者的混合卵囊人工感染雏鸡，观察感染前、后雏鸡体质量变化和排出卵囊减少量，评估其对多种球虫感染的免疫保护效果。[结果]EaUCE基因开放阅读框为444 bp，编码148个氨基酸，BLAST分析显示，柔嫩艾美耳球虫、堆型艾美耳球虫、巨型艾美耳球虫UCE氨基酸序列同源性均大于95%。pVAX-EaUCE能够在鸡体内成功转录和表达。pVAX-EaUCE免疫后，鸡体的CD4+/CD3+和CD8+/CD3+细胞比例显著升高，IFN-γ、IL-2、IL-4、TNFSF15、IL-17D、TGF-β4 mRNA和IgY抗体水平显著升高，表明免疫该质粒能诱导细胞和体液产生免疫反应（P<0.05）。pVAX-EaUCE免疫后，与对照组相比，免疫组鸡体质量显著增加，卵囊产量明显下降，肠道损伤也相应减轻。[结论]鸡球虫共同抗原UCE具有良好的免疫原性，可对多种球虫感染提供部分免疫保护力，有望成为抵抗多种球虫感染的候选抗原。
[Objectives] Ubiquitin-conjugating enzyme domain-containing protein(UCE)is one of the common antigens among Eimeria tenella, E.acervulina, and E.maxima identified in our previous study. The present study was performed to further evaluate its immunogenicity and protective efficacy against infections of multiple Eimeria species in chickens. [Methods] EaUCE gene was amplified from the total RNA of sporulated oocysts of E.acervulina and subcloned into eukaryotic expression vector pVAX1. The transcription and expression of pVAX-EaUCE in the injected muscles were detected by RT-PCR and Western blot in vaccinated chickens. Cellular and humoral immune responses induced by pVAX-EaUCE vaccination were detected by flow cytometry and indirect ELISA. The protective efficacy of pVAX-EaUCE was evaluated by vaccination and challenge with E.tenella, E.acervulina, E.maxima and mixed infection of the three Eimeria species respectively. [Results] The length of the open reading frame(ORF)of EaUCE was 444 nucleotides which encoding 148 amino acids. BLAST analysis revealed that UCE shared similarities of beyond 95% in amino acid sequences among E.tenella, E.acervulina and E.maxima. The pVAX-EaUCE was successfully transcribed and expressed in the injected muscle, the proportion of CD4+/CD3+ and CD8+/CD3+ T lymphocytes, IFN-γ, IL-2, IL-4, TNFSF15, IL-17D, TGF-β4 mRNA and IgY level increased significantly compared to the controls, which suggested that pVAX-EaUCE vaccination could significantly induce the cellular and humoral immune responses in chickens. Vaccination with pVAX-EaUCE significantly increased the weight gains, decreased the oocyst productions and alleviated the enteric lesions compared to he controls, which suggested that pVAX-EaUCE provided partially protection against multiple Eimeria species in chickens. [Conclusions] The coccidial common antigen of UCE