摘要
通过RT-PCR技术从刺激的藏獒犬外周血淋巴细胞中成功克隆白介素18基因全长,其大小为582bp,编码194个氨基酸。与GenBank上发表的犬IL-18(NM001003305)比较,序列的同源性为100%,与犬、猫、猪,牛、羊、人的IL-18基因核苷酸同源性分别为100%、91.2%、91.1%、88.5%、89.3%、84.4%。系统进化树可以看出,藏獒犬基因与犬的亲缘关系最近,与猫的其次,与人是较远的。然后构建-IL18去信号肽的原核表达载体pET-30a-IL18,转化BL2L,IPTG诱导,表达产物经SDS-PAGE分析表明,表达出25kd融合蛋白而且目的蛋白主要以包涵体的形式存在,westbloting验证表达的蛋白正确。
The Canine IL-18 gene was amplified by RT-PCR from the total RNA of Canine peripheral blood leucocyte in- duced with LPS and Con A. The cloned IL-18 gene was 582 bp in length encoding a polypetide of 193 anino acid. Compared with the gene of canine, Felis eatus, Sus scrofa, Bos taurus, Ovis aries and Human on GenBank, respectively, the nucleotide homology showed 100% ,91.2% ,91.1% .88.5% .89.3% .84.4%. The phylogenetic tree analysis showed that genetic relationship was most recently between canine, by turns Felis catus, Susscrofa. Then the recombinant expression plasmid（pET-30a-IL18） was constructed and induced by IPTG. SDS-PAGE analysis showed that the fusion protein with a molecular weight of 25ku ,esisted in include body. Expression protein was detected by westernbloting.
作者
宋世斌
胡永浩
何强
敬淑燕
仝保全
Song Shi-bin;Hu Yong-hao;He Qiang;Jing Shu-yan;Tong Bao-quan(Gansu Agricultural University College of Veterinary Medicine, lanzhu, gansu 730070,China;Gansu Agriculture Technology College;Lanzhou Native Mastiff Park)
出处
《畜牧兽医杂志》
2018年第4期4-8,共5页
Journal of Animal Science and Veterinary Medicine
基金
甘肃省高等学校科研项目资助(2015A-195)
关键词
藏獒犬
白介素18
原核表达
Zangao Canine
IL-18
Prokaryotic Expression
