猪干扰素IFNE1基因克隆及重组表达载体的构建  

Clone and Construction of Recombinant Expression Vector of Porcine IFNE1

作　　者：台玉磊[1] 王艳玲[1] 王伟杰[1] 韩立强[1] 张志强[1] 臧猛[1] 王静[1] 杨国宇[1] 

机构地区：[1]河南农业大学动物生理生化实验室,郑州450002

出　　处：《中国农学通报》2008年第11期60-64,共5页Chinese Agricultural Science Bulletin

基　　金：国家自然科学基金(30671531)。

摘　　要：依据电子延伸序列设计一对克隆引物,用RT-PCR法从猪胃组织扩增出猪干扰素epsilon 1(IFNE1)基因的完整编码区并进行序列分析;再根据克隆的序列设计一对表达引物,用PCR法从重组克隆载体中扩增出含EcoRI/XhoI酶切位点的猪IFNE1片段,插入原核表达载体,转化至宿主菌,诱导表达,SDS-PAGE鉴定融合蛋白。结果表明,克隆的猪IFNE1基因包含完整的开放阅读框架,长为586bp,ORF为582bp,编码193个氨基酸,与人、小鼠的同源性分别为83.6%和69.2%,推测的氨基酸序列与人、小鼠的同源性分别为76.2%和55.2%,表达的融合蛋白分子量约为47kD。A pair of cloning primers was designed according to the cloning principle of in silico sequence. The whole coding region of interferon epsilonl （IFNE1） extracted from mucosal tissue of porcine stomach was amplified by RT-PCR, and sequence analyzed. Then, a pair of expression primers was designed according to the cloned sequence. A fragment of porcine IFNE1 cDNA containing EcoRI/XhoI was amplified from recombinant vector by PCR. The fragment was to construct a recombinant expression vector. Subsequently, the recombinant vector was transformed into parasitic bacterium. The fusion protein, which was induced was confirmed by SDS-PAGE. The results showed that the cloned sequence contain the open reading frame of 582 bp and encodes 193 amino acids. Identity analysis showed that the porcine IFNE1 nucleotide sequence shared 83.6% and 69.2% homology with that of human, mouse, the predicted amino acid shared 76.2% and 55.2% homology with that of human, mouse. The fusion protein expressed in E.coli BL21（DE3） was about 47 kD and mainly existed as inclusion bodies.

关 键 词：猪 干扰素ε1 克隆 序列分析 原核表达 

分 类 号：S859.797[农业科学—临床兽医学] Q786[农业科学—兽医学]