新城疫病毒Roakin株HN基因的克隆及真核表达载体的构建  

Cloning and Construction of Eukaryotic Expression Vector of HN Gene of Newcastle Disease Virus Roakin Strain

作　　者：张继乐[1,2] 陈豪泰[2] 丁耀忠[2] 孙德惠[2] 马丽娜[2] 张杰[2] 金雷[2] 高云英[1] 刘永生[2] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,农业部畜禽病毒学重点开放实验室,甘肃兰州730046

出　　处：《西北农业学报》2009年第2期19-23,37,共6页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：中国农业科学院兰州兽医研究所所长基金项目(2007-13)

摘　　要：克隆新城疫病毒(Newcastle disease virus,NDV)Roakin株HN基因,并构建真核表达载体。应用RT-PCR扩增出NDV Roakin株HN基因,将其克隆入pMD18-T载体,并进行了测序鉴定和HN基因的序列分析。然后分别将NDV的HN基因和选择标记基因—二氢叶酸还原酶(dhfr)基因插入到真核表达载体pCI-neo中,通过PCR扩增、酶切分析和测序分析等鉴定所构建的重组真核表达载体。RT-PCR扩增的NDVRoakin株HN序列与GenBank中标准毒株(登录号AY289000)HN序列同源性为99.8%,构建了HN基因的真核表达质粒pCI-HN-D。成功构建了含有NDV Roakin株HN基因和dhfr基因的真核表达载体。To clone The HN gene of Newcastle disease virus（NDV） Roakin strain and construct a eukaryotic expression vector which can be expressed in Chinese Hamster Ovary Cells CHO/dhfr-.The HN gene of Newcastle disease virus（NDV） Roakin strain was amplified by RT-PCR and cloned into pMD18-T vector and sequenced.The HN gene of the virus and dhfr gene was cloned into high efficient eukaryotic vector pCI-neo.PCR amplification,restriction emzyme digestion and sequencing analysis was applied to identify the recombinant expression plasmids.The sequence homology of HN cloned with the HN（AY289000）on GenBank was 99.8%,Mammalian recombinant expression plasmid pCI-HN-D was constructed.Recombinant expression plasmid pCI-HN-D harboring dhfr gene and HN gene of NDV has been successfully constructed and lay the foundation of high-level expression of HN protein of NDV in mammalian cell CHO/dhfr-.

关 键 词：新城疫病毒 HN基因 dhfr基因 真核表达载体 

分 类 号：S851.347.201[农业科学—预防兽医学]