延边黄牛白细胞介素18基因的克隆及序列分析

本试验旨在扩增延边黄牛白细胞介素18(IL-18)全长cDNA,并对其序列进行进化分析。根据GenBank中已公布的牛IL-18 cDNA序列设计1对引物,利用RT-PCR从刀豆蛋白(ConA)和植物分裂素(PHA)双重刺激36 h活化的延边黄牛脾淋巴细胞中扩增出牛IL-18全长cDNA,进行PCR、酶切和测序鉴定,并做进化分析及二级结构预测。扩增出延边黄牛IL-18全长cDNA,大小为760 bp,其中有两个氨基酸突变。进化分析结果显示,延边黄牛IL-18与牛、山羊、绵羊、马和猪的同源性较高,均大于90%,与同种群牛的同源性最高,而与原鸡的同源性最低,为51.1%。本研究确定了IL-18的种群差异,并预测了二级结构特性,为进一步研究延边黄牛IL-18的基因表达、生物活性及其作为佐剂的应用奠定了基础。

Cloning and Sequence Analysis of Yanbian Bovine Interleukin 18

The aim of this paper was to amplify full length cDNA genes of Yanbian BoIL-18 and make phylogenetic anylysis.Designing and synthesizing one pair of primers according to bovine IL-18 gene sequences published in GenBank.Bovine IL-18 gene was amplified by RT-PCR from bovine splenic lymphocyte which stimulating by both ConA and PHA for 36 h,then was cloned,double restriction digested and sequenced to identify the positive samples,further to carry out phylogenetic analysis and secondary structure prediction.Having amplified full length cDNA genes of 760 bp of Yanbian BoIL-18,there were two amino acid different.Phylogenetic analysis showed that Yanbian BoIL-18 had high homology with Bos taurus,Capra hircus,Ovis aries,Equus caballus,Sus scrofa,Canis lupus and had low homology with Gallus gallus(51.1%).We make sure the group differences of IL-18 and predicted the secondary structure,laying a foundation for further expressing bovine IL-18,biological activity and utilized as a adjuvant.