猪繁殖与呼吸综合征病毒兰州分离株ORF3基因的克隆及杆状病毒载体的构建  

Cloning of ORF3 Gene of PRRS Virus and Construction of Its Baculovirus Epression Vector

作　　者：赵娜[1,2] 田宏[1] 祁燕蓉[2] 满自萍[1,2] 吴锦艳[1] 尚佑军[1] 何生虎[2] 刘湘涛[1] 

机构地区：[1]中国农业科学院兰州兽医研究所,国家口蹄疫参考实验室,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 [2]宁夏大学农学院,宁夏银川750021

出　　处：《西北农业学报》2009年第3期1-5,共5页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家“十一五”高技术研究发展计划(863)项目(2006AA10A207);国家“十一五”科技支撑计划课题(2006BAD06A11)

摘　　要：根据猪繁殖与呼吸综合症病毒已知序列(ch-1a,AY032626)设计包含完整ORF3序列的1对引物,采用RT-PCR方法扩增PRRSV甘肃Lanzhou株的ORF3基因,将其克隆到杆状病毒载体pFastBac HTA上,经酶切鉴定、PCR鉴定筛选出阳性重组转移载体pFastBac HTA-ORF3,并对阳性质粒进行测序及序列分析;将pFastBac HTA-ORF3转化到DH10Bac感受态细胞中,与Bacmid发生位点特异性转座作用,获得重组转座子rBacmid-ORF3。成功构建了PRRSV Lanzhou株ORF3基因的杆状病毒载体,为基因工程疫苗的研制奠定了基础。According to the published genome sequences of PRRSV（ch-1a）,one pair of specific primers were designed to amplify ORF3 gene of Lanzhou strain of Gansu province by RT-PCR. The amplified fragment was cloned into pFastBacHTA donor plasmid. The recombinant pFastBacHTA was identified by digestion with endonuclease enzyme, PCR amplification and sequencing. Then the purified plasmid was transformed into DH10Bac-host cells,producing specific transposition with Bacmid to yield the recombinant shuttle vehicles, rBacmid-ORF3. The baculovirus epression vector was successfully constructed, which would be useful for production of gene vaccination in future.

关 键 词：猪繁殖与呼吸综合征病毒 ORF3基因 克隆 杆状病毒表达载体 

分 类 号：S858.28[农业科学—临床兽医学]