柔嫩艾美耳球虫棒状体颈部蛋白EtRON2基因的克隆及序列分析

Cloning and Sequence Analysis of Eimeria tenella Rhoptry Neck Protein EtRON2 Gene

预测并克隆柔嫩艾美耳球虫棒状体颈部蛋白2(EtRON2)基因,分析其结构特点及与顶复门其他虫属间的差异。本研究利用生物信息学和比较基因组学技术注释拼接得到柔嫩艾美耳球虫(Eimeria tenella)棒状体颈部蛋白2(EtRON2)的基因序列,用RT-PCR方法扩增出Etron2全长ORF;以E.tenella子孢子的基因组DNA为模板,用PCR方法成功扩增出Etron2ORF片段的gDNA序列片段。生物信息学分析结果显示,Etron2ORF片段的gDNA含有11个内含子,ORF全长为4 020bp,编码一段全长为1 339个氨基酸的多肽片段,与其他物种RON2的氨基酸序列相似性为15%～41%;Etron2基因在顶复门原虫中相对保守,与犬新孢子虫及弓形虫相似性最高,亲缘关系最近,Etron2的成功克隆填补了柔嫩艾美耳球虫在此方面研究的空白,为进一步研究其功能打下了基础。

To predict and clone Eirneria tenella rhoptry neck protein 2 EtRON2 gene, and analysis the structure character of Etron2. The sequence encoding E. tenella RON2 （EtRON2） protein were predicted from the crow date of Eimeria tenella gene sequences using the bioinformatics tool, amplified by RT-PCR from E. tenella （Guangdong strain） total RNA template. The genome DNA sequence transcripted this ORF was obtained by PCR from E. tenella （Guangdong strain） DNA template, using the gene specific primer based on the cloned EtRON20RF. This gene includies 11 introns, and is 5 197 bp in tenth, the ORF is 4 020 bp in full lenth, and coding a peptide of 1 339 aa in lenth with a signal peptide sequence. Amino acid sequences alignment of EtRON2 with those from other several apicomplexan parasites by Clustal W showed EtRON2 shared 15%-41 % amino acid identities with other RON2s. These results indicated that the Apicomplex has conserved ron2 gene sequence, comparison of other Apicomplex, the Etron2 were phylo- genetically most closely related to Neospora caninurn and Toxoplasrna gondii （41% Tgron2 gene sequence similarity）, thereby demonstrating their high level of genealogical relatedness.