Zhu Huili, Yan Ruofeng, Wang Song, Song Xiaokai, Xu Lixin, Li Xiangrui
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu, PR China.
Mol Biochem Parasitol. 2012 Nov;186(1):21-8. doi: 10.1016/j.molbiopara.2012.09.002. Epub 2012 Sep 17.
Eimeria acervulina (E. acervulina) is one of the seven species of Eimeria infected in chicken. Until now, only a few antigen genes of E. acervulina have been reported. In this study, a cDNA expression library of E. acervulina sporozoites was constructed in a eukaryotic expression vector, pVAX1.0. Subsequently, the library was divided into pools and inoculated into chickens to observe the ability of the antigens to induce humoral immune response and cell-mediated immune response. The positive pools that stimulated significant immune responses were fractionated sequentially until a single positive clone was screened. After three rounds of screening, a clone, named as cSZ-JN1, with the ability to stimulate chicken immune response was obtained. The sequence analysis showed that the opening reading fragment (ORF) of cSZ-JN1 was 615 bp in size and encoded a predicted protein of 204 amino acids with 21.8 kDa. BLASTN and other sequence databases searches revealed that the identity of the amino acid sequence of cSZ-JN1 to the complete sequence of Eimeria tenella annotated protein (ETH_00022005.1.pep) was 31.37% and to Toxoplasma gondii ME49 hypothetical protein (gb|EEB00972.1|) 24%, and had no significant homology with the known genes of E. acervulina and other parasites. Immunofluorescence analysis using antibody against recombinant cSZ-JN1 indicated that this protein was expressed in sporozoite and merozoite development stages. Animal challenge experiments demonstrated that the recombinant protein of cSZ-JN1 and DNA vaccine carrying cSZ-JN1 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens and presented anti-coccidial index more than 160. All the above results suggested that the cSZ-JN1 was a novel E. acervulina antigen and could be an effective candidate for the development of new vaccine against E. acervulina infection.
堆型艾美耳球虫（E. acervulina）是感染鸡的七种艾美耳球虫之一。到目前为止，仅报道了少数堆型艾美耳球虫的抗原基因。在本研究中，在真核表达载体pVAX1.0中构建了堆型艾美耳球虫子孢子的cDNA表达文库。随后，将文库分成池并接种到鸡中，以观察抗原诱导体液免疫应答和细胞介导免疫应答的能力。刺激显著免疫应答的阳性池依次分级分离，直至筛选出单个阳性克隆。经过三轮筛选，获得了一个具有刺激鸡免疫应答能力的克隆，命名为cSZ-JN1。序列分析表明，cSZ-JN1的开放阅读框（ORF）大小为615 bp，编码一个预测的204个氨基酸的蛋白质，分子量为21.8 kDa。BLASTN和其他序列数据库搜索显示，cSZ-JN1的氨基酸序列与注释的柔嫩艾美耳球虫蛋白质（ETH_00022005.1.pep）的完整序列的同一性为31.37%，与刚地弓形虫ME49假设蛋白质（gb|EEB00972.1|）的同一性为24%，与堆型艾美耳球虫和其他寄生虫的已知基因无显著同源性。使用抗重组cSZ-JN1抗体的免疫荧光分析表明，该蛋白在子孢子和裂殖子发育阶段表达。动物攻毒实验表明，cSZ-JN1的重组蛋白和携带cSZ-JN1的DNA疫苗可显著增加免疫鸡的平均体重增加量，降低平均病变评分和卵囊产量，抗球虫指数超过160。上述所有结果表明，cSZ-JN1是一种新型的堆型艾美耳球虫抗原，可能是开发抗堆型艾美耳球虫感染新疫苗的有效候选物。
