Zhang ZhenChao, Liu LianRui, Huang JingWei, Wang Shuai, Lu MingMin, Song XiaoKai, Xu LiXin, Yan RuoFeng, Li XiangRui
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China.
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China.
Vet Parasitol. 2016 Jan 15;215:96-105. doi: 10.1016/j.vetpar.2015.10.028. Epub 2015 Oct 31.
Eimeria acervulina (E. acervulina) is an intestinal protozoon and few of its antigen genes were researched. In this study, the gene of E. acervulina microneme protein 2 (EaMIC2) was cloned and characterized. The degenerate primers for the conserved sequence of microneme protein 2 were designed based on Eimeria tenella microneme 2 (EtMIC2) (AF111839.1), Eimeria maxima microneme 2 (EmMIC2) (FR718971.1) and E. brunetti microneme 2 (EbMIC2) (AB723700.1) to amplify the conserved sequence of microneme protein 2. According to the conserved sequence, specific primers for the rapid amplification of cDNA ends (RACE) were designed to amplify the 3'- and 5'-ends of EaMIC2. The full length cDNA of this gene was obtained by overlapping the sequences of 3'- and 5'-extremities and amplification by reverse transcription PCR. The sequence analysis revealed that the opening reading frame (ORF) of EaMIC2 was 882bp and encoded a protein of 293 amino acids with 29.81kDa. The most part of the mature protein (base: 82-882bp, amino acid: 28-293aa) of EaMIC2 (mpmEaMIC2) was inserted into pCold TF to produce recombinant mpmEaMIC2. Western blotting assay was used to analysis the immunogenicity of mpmEaMIC2 and the result showed that the recombinant protein was successfully recognized by the sera of chickens experimentally infected with E. acervulina, while the native protein in the somatic extract of sporozoites was as well detected by sera from rats immunized with the recombinant protein of mpmEaMIC2. Immunofluorescence analysis using antibody against recombinant protein mpmEaMIC2 indicated that this protein was expressed in the sporozoites and merozoites stages of E. acervulina. Animal challenge experiments demonstrated that the recombinant protein of mpmEaMIC2 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens. All the above results suggested that the EaMIC2 was a novel E. acervulina antigen and could be an effective candidate for the development of new vaccine against this parasite.
堆型艾美耳球虫（E. acervulina）是一种肠道原生动物，其抗原基因鲜有研究。本研究对堆型艾美耳球虫微线体蛋白2（EaMIC2）基因进行了克隆和鉴定。基于柔嫩艾美耳球虫微线体2（EtMIC2）（AF111839.1）、巨型艾美耳球虫微线体2（EmMIC2）（FR718971.1）和布氏艾美耳球虫微线体2（EbMIC2）（AB723700.1）设计了微线体蛋白2保守序列的简并引物，以扩增微线体蛋白2的保守序列。根据保守序列，设计了用于快速扩增cDNA末端（RACE）的特异性引物，以扩增EaMIC2的3'端和5'端。通过重叠3'端和5'端序列并进行逆转录PCR扩增，获得了该基因的全长cDNA。序列分析表明，EaMIC2的开放阅读框（ORF）为882bp，编码一个293个氨基酸、分子量为29.81kDa的蛋白质。将EaMIC2成熟蛋白的大部分（碱基：82 - 882bp，氨基酸：28 - 293aa）（mpmEaMIC2）插入pCold TF中以产生重组mpmEaMIC2。采用蛋白质免疫印迹法分析mpmEaMIC2的免疫原性，结果显示该重组蛋白能被实验感染堆型艾美耳球虫的鸡血清成功识别，而用mpmEaMIC2重组蛋白免疫的大鼠血清也能检测到子孢子体细胞提取物中的天然蛋白。使用抗重组蛋白mpmEaMIC2的抗体进行免疫荧光分析表明，该蛋白在堆型艾美耳球虫的子孢子和裂殖子阶段表达。动物攻毒实验表明，mpmEaMIC2重组蛋白可显著提高免疫鸡的平均体重增加量，降低平均病变评分和卵囊产量。上述所有结果表明，EaMIC2是一种新型的堆型艾美耳球虫抗原，可能是开发针对该寄生虫新疫苗的有效候选物。
