牦牛α-干扰素基因的克隆及其原核表达的研究  

Cloning and expression of yak interferon-α gene in Escherichia coli

作　　者：景志忠[1] 李玉萍[1] 窦永喜[1] 陈国华[1] 房永祥[1] 才学鹏[1] 

机构地区：[1]中国农业科学院兰州兽医研究所甘肃省动物寄生虫病重点实验室/家畜疾病病原生物学国家重点实验室,甘肃兰州730046

出　　处：《中国预防兽医学报》2008年第3期194-199,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：甘肃省自然基金项目(3ZS061-A25-081);国家“863”计划(2006AA10A203)

摘　　要：植物血凝素(PHA)诱导培养牦牛外周血淋巴细胞,提取总RNA,采用RT-PCR技术扩增牦牛IFN-α-A和IFN-α-Ⅱ基因,分别经PCR、酶切鉴定及测序分析后,再分别构建原核表达重组质粒,用SDS-PAGE和Western blot分析IPTG诱导表达的重组蛋白。序列分析表明,牦牛IFN-α-A基因ORF为570 bp,与NCBI上发表的奶牛IFN-α-A参考序列同源性为94.4%,与黄牛IFN-α-B基因(M10953)的同源性最高,为97.0%;牦牛IFN-α-Ⅱ基因ORF为588 bp,与NCBI上发表的奶牛IFN-α-Ⅱ参考序列同源性为94.2%,与黄牛IFN-δ-1基因(XM-871297)的同源性最高,为96.1%。分子遗传进化树分析表明牦牛IFN-α-A和IFN-α-Ⅱ与黄牛、奶牛和水牛亲缘关系最近,与猪、马、人的次之,与其它动物都较远,说明这两基因均具有种属差异性。诱导表达重组蛋白分析表明,pGEX4T/IFN-α-A和pGEX4T/IFN-α-Ⅱ在大肠杆菌中得到了正确表达,均可表达约44 ku的融合蛋白,主要以包涵体形式存在,且具有与抗血清发生反应的生物活性。Total RNA was isolated from yak peripheral blood lymphocytes stimulated with PHA. The yak interferon-α-A （IFN- α-A） and the yak interferon- α - Ⅱ （IFN- α- Ⅱ ） cDNAs were amplified by RT-PCR, and sequenced. The yak IFN- α-A gene contained a ORF of 570 bp in length, which shared 94.4 % sequence homology with the cow IFN-α-A gene and 97.0 % with cattle IFN-α-B（M10953） gene. The yak IFN-α-Ⅱ gene had a ORF of 588 bp in length, and shared 94.2 % and 96.1% sequence identities with that of cow and cattle IFN-δ-1 （XM-871297）. Phylogenetic analysis showed that the yak IFN-α-A and IFN-α-Ⅱ genes were both species-specific. The yak IFN-α-A and the IFN-α- Ⅱ were cloned into vector pGEX4T-1, respectively, and expressed in BH21 E.coli cells by IPTG induction. SDS-PAGE analysis showed that both fusion proteins of about 44 ku were expressed in the form of inclusion bodies. Western blot analysis showed that yak IFN- α -A and IFN- α- Ⅱ were both expressed with specific antigenic activities.

关 键 词：牦牛IFN-α-A基因 牦牛IFN-α-Ⅱ基因 分子克隆 遗传演化关系 表达分析 

分 类 号：Q785[生物学—分子生物学]