摘要
提取经植物血凝素(PHA)诱导培养的牦牛外周血淋巴细胞总RNA,采用RT-PCR技术扩增并克隆了牦牛IFN-β基因,经PCR和酶切鉴定及测序分析,再克隆到pGEX-4T-1质粒载体中,构建了原核表达重组载体,经IPTG诱导表达重组蛋白,可表达约41 ku的牦牛IFN-β融合蛋白,主要以包涵体形式存在。序列分析结果表明,牦牛IFN-β基因ORF为498 bp,与乳牛IFN-β参考序列的同源性高达98.6%,与猪、马、猫、人IFN-β基因的同源性分别为72.5%、68.9%、66.1%和63.5%,而与狗、鼠、鸡等动物的同源性均不超过25%;分子遗传进化树分析也表明,牦牛与乳牛IFN-β基因的亲缘关系最近,而与鼠、狗、鸡等的亲缘关系较远,说明IFN-β基因有种属差异性。
Total RNA was isolated from yak peripheral blood lymphocytes stimulated with PHA. The yak interferon-β （IFN-β）cDNA was amplified by RT-PCR, and then cloned and sequenced. After being sequenced, the sub-cloned IFN-β gene was inserted successfully to the expression vector pGEX-4T-1, then stimulated with IPTG to express recombinant protein. SDS-PAGE analysis showed that the fusion protein was approximately 41 ku in size in the form of inclusion bodies. The ORF of the cloned yak IFN-β gene was 498 bp in length, and the homologies between the yak IFN-β gene and the IFN-β genes of cow, pig, horse, cat and human were 98.6%,72.5%, 68.9%, 66.1% and 63.5%, respectively, while the homologies between the yak IFN-β gene and the IFN-β genes of mice, dog and chicken were all less than 25%. Phylogenetic analysis based on the amino acid sequence from the IFN-β gene showed that the yak IFN-β gene was species-specific.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2006年第1期40-45,共6页
Chinese Veterinary Science
基金
农业结构调整重大技术研究专项项目(04-10-03B)
兰州市科技攻关计划项目(05-1-47)
