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中国农学通报

中国农学通报 ›› 2008, Vol. 24 ›› Issue (11): 60-64.doi: 10.11924/j.issn.1000-6850.2008-0460

所属专题： 生物技术； 畜牧兽医

• 生物技术科学 • 上一篇    下一篇

猪干扰素IFNE1基因克隆及重组表达载体的构建

台玉磊,王艳玲,王伟杰,韩立强,张志强,臧 猛,王 静,杨国宇   

  • 收稿日期:2008-07-22 修回日期:2008-08-15 出版日期:2008-11-05 发布日期:2008-11-05

Clone and construcion of recombinant expression vector of Porcine IFNE1

Tai Yulei, Wang Yanling, Wang Weijie, Han Liqiang, Zhang Zhiqiang, Zang Meng,Wang Jing, Yang Guoyu   

  • Received:2008-07-22 Revised:2008-08-15 Published:2008-11-05 Online:2008-11-05

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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586 bp，ORF为582 bp，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47 kDa。

Abstract: A pair of cloning primers were designed according to the cloning principle of in silico sequence. The whole coding region of interferon epsilon1(IFNE1) extracted from mucosal tissue of porcine stomach was amplified by RT-PCR, and sequence analyzed. Then, a pair of expression primers were designed according to the cloned sequence. A fragment of porcine IFNE1 cDNA containing EcoRI/XhoI were amplified from recombinant vector by PCR. The fragment was to construct a recombinant expression vector. Subsequently, the recombinant vector were 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 kDa and mainly existed as inclusion bodies.