Molecular Characterization and Immune Protection of a New Conserved Hypothetical Protein of Eimeria tenella.
Molecular Characterization and Immune Protection of a New Conserved Hypothetical Protein of Eimeria tenella.
复制标题
柔嫩艾美耳球虫新保守假设蛋白的分子表征和免疫保护
DOI：
10.1371/journal.pone.0157678
复制
发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Han H
中科院分区：
文献类型：
--
作者：
Zhai Q;Huang B;Dong H;Zhao Q;Zhu S;Liang S;Li S;Yang S;Han H
柔嫩艾美耳球虫的基因组序列已被测序，但这些基因中有70%以上目前被归类为具有未知功能或被注释为保守的假设蛋白，其中很少有人被研究。本研究中，E.基于表达序列标签（EST），利用cDNA 5 '端快速扩增（5' RACE）技术克隆了tenella，命名为EtCHP 559。EtCHP 559全长cDNA为1746 bp，包含一个1224 bp的开放阅读框（ORF），编码一个407个氨基酸的多肽，预测分子量为46.04 kDa。实时定量PCR分析表明，EtCHP 559在子孢子中的表达水平高于其他发育阶段（未孢子化卵囊，孢子化卵囊和第二代裂殖子）。将ORF插入pCold-TF中以产生重组EtCHP 559。Western blotting结果表明，重组蛋白能被兔抗E.柔嫩孢子EtCHP 559抗体免疫定位结果表明，EtCHP 559主要分布在游离子孢子的寄生虫表面，在完全培养基中孵育后，EtCHP 559开始集中在寄生虫的前部区域。EtCHP 559变得均匀地分散在未成熟和成熟的基质中。使用抗rEtCHP 559多克隆抗体抑制EtCHP 559功能降低了E. tenella子孢子侵入宿主细胞> 70%。动物攻毒试验表明，重组EtCHP 559能显著提高感染鸡的平均增重，减少卵囊排出量，减轻盲肠病变，抗球虫指数>160。tenella。这些结果表明，EtCHP 559在子孢子侵入中起重要作用，并且可能是开发针对E. tenella。
The genome sequences of Eimeria tenella have been sequenced, but >70% of these genes are currently categorized as having an unknown function or annotated as conserved hypothetical proteins, and few of them have been studied. In the present study, a conserved hypothetical protein gene of E. tenella, designated EtCHP559, was cloned using rapid amplification of cDNA 5'-ends (5'RACE) based on the expressed sequence tag (EST). The 1746-bp full-length cDNA of EtCHP559 contained a 1224-bp open reading frame (ORF) that encoded a 407-amino acid polypeptide with the predicted molecular weight of 46.04 kDa. Real-time quantitative PCR analysis revealed that EtCHP559 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second generation merozoites). The ORF was inserted into pCold-TF to produce recombinant EtCHP559. Using western blotting, the recombinant protein was successfully recognized by rabbit serum against E. tenella sporozoites. Immunolocalization by using EtCHP559 antibody showed that EtCHP559 was mainly distributed on the parasite surface in free sporozoites and became concentrated in the anterior region after sporozoites were incubated in complete medium. The EtCHP559 became uniformly dispersed in immature and mature schizonts. Inhibition of EtCHP559 function using anti-rEtCHP559 polyclonal antibody reduced the ability of E. tenella sporozoites to invade host cells by >70%. Animal challenge experiments demonstrated that the recombinant EtCHP559 significantly increased the average body weight gain, reduced the oocyst outputs, alleviated cecal lesions of the infected chickens, and resulted in anticoccidial index >160 against E. tenella. These results suggest that EtCHP559 plays an important role in sporozoite invasion and could be an effective candidate for the development of a new vaccine against E. tenella.