柔嫩艾美耳球虫新基因的生物信息学分析与克隆表达

Bioinformatics Analysis,Cloning and Expression of a Novel Gene from Eimeria tenella

为了研究柔嫩艾美耳球虫(Eimeria tenella)的两个主要入侵发育阶段——子孢子和裂殖子的差异基因,根据已报道的子孢子与裂殖子差异的ESTs序列,应用RACE技术克隆获得了子孢子阶段差异表达的新基因全长cDNA序列,命名为ZL583。该基因全长为862 bp,开放阅读框(ORF)为486 bp,编码161个氨基酸,编码蛋白的分子量约为16.9 kD,利用生物信息学分析软件,分析新基因所编码蛋白的性质、定位、结构等特征。利用荧光定量PCR对柔嫩艾美耳球虫不同发育阶段该基因的表达量进行分析显示,子孢子阶段的表达高于其他发育阶段。将ZL583克隆于pET-28a中构建重组质粒,转化大肠杆菌BL21(DE3)后经IPTG诱导表达,获得重组蛋白分子量约23 kD。免疫印迹分析显示该重组蛋白可与兔抗子孢子血清发生特异性反应,表明该蛋白具有较好的反应原性。该结果为进一步研究该基因的生物学功能奠定基础。

In order to study the different genes between the invasion stages of Eimeria tenella,expressed sequence tags（ESTs）of sporoziotes and merozoites were analyzed and a novel gene named ZL583 was identified by rapid amplification of cDNA ends（RACE）approaches.The full-length cDNA was 862 bp with an open reading frame（ORF）of 486 bp encoded a polypeptide of 161 amino acids with the predicted molecular weight of 16.9 kD.Bioinformatics methods were used to predict the structure and function of the gene.Real-time quantitative PCR analysis revealed that the expression level of ZL583 in sporozoites is higher than those in the other developed stages（unsporulated oocysts,sporulated oocysts and second-generation merozoites）.The sequence encoding the mature protein was amplified by PCR,cloned into the pET-28a vector and expressed in Escherichia coli BL21（DE3）.SDS-PAGE indicated that the recombinant protein was 23 kD and soluble.Western blotting revealed that the recombinant protein could be specifically recognized by polyclonal antibodies against sporozoites of E.tenella.These results would in favour of studying the biological function of the gene.