柔嫩艾美耳球虫BJ株Et1A基因的克隆及序列分析

Clone and Sequence Analysis of Gene Et1A of Eimeria tenella BJ Strain

本文参照已发表的柔嫩艾美耳球虫(E.tenella)折光体蛋白基因序列,根据其阅读框及载体序列特点,两端分别加上EcoRⅠ和HindⅢ酶切位点设计引物,以E.tenellaBJ株的总RNA为模板,进行RT-PCR扩增,成功扩增出Et1A基因。将PCR产物与pGEM-T载体连接后,转化JM109感受态细胞,蓝白斑法筛选阳性克隆并测序。测序表明,该基因全长1978bp,其阅读框大小为1944bp;与GenBank登记的Et1A基因相比较,有6个部位、共7个碱基发生了突变,其中5个有意义突变,2个无意义突变,同源性达99 5%。与堆型艾美耳球虫(E.acervulina)的Ea1A基因的同源性为79 3%,而与牛艾美耳球虫(E.bovis)的同源性只有19 5%。表达产物具有核苷酸转氢酶的功能,推测可为子孢子侵入宿主细胞提供能量。

In order to clone the gene Et1A,the total RNA of Eimeria tenella BJ Strain was extracted and used as template of RTPCR.The primers was designed according to the published database.It involved an EcoR Ⅰ or Hind Ⅲ cleavage site respectively in order to be ligated to the plasmid.After RTPCR,the cloned Et1A was ligated to the vector pGEMT and transformated JM109 competent cell.The positive colones were used to determine its nucleotide sequence.The results indicated that,the fulllength of Et1A was 1978 bp in E.tenella BJ Strain,which had an ORF of 1944 bp.Compared with the Et1A1 database of GenBank,mutation occurred at 7 bases of 6 regions and the homology was 995%.Et1A and Ea1A had a 793% identity.The derived protein of Et1A is functional similarity to nucleotide transhydrogenase.It is a speculated energy support during the invasion of sporozoites into host cells.