PRRSV兰州分离株ORF3和ORF5基因的克隆与序列分析及其杆状病毒表达载体的构建  

Cloning and sequence analysis of ORF3 and ORF5 genes of PRRSV Lanzhou strain and construction of their baculovirus expression vectors

作　　者：赵娜[1,2] 田宏[1] 吴锦艳[1] 祁燕蓉[2] 满自萍[1,2] 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：《中国兽医科学》2009年第3期189-195,共7页Chinese Veterinary Science

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”科技支撑计划项目(2006BAD06A11)

摘　　要：根据GenBank中猪生殖与呼吸综合征病毒（PRRSV）中国代表株CH-1a的基因组序列，设计包含完整ORF3、ORF5基因序列的2对特异引物，利用RT-PCR技术扩增PRRSV兰州分离株（Lanzhou株）的ORF3和ORF5基因，并对其进行序列测定，同时与已发表的13株PRRSV进行同源性比较。将扩增的PRRSV Lanzhou株ORF3、ORF5基因分别克隆到杆状病毒表达载体pFastBacHTA上，将经酶切及测序鉴定的阳性重组质粒pFastBacHTA-ORF3、pFastBacHTA-ORF5分别转化到DH10Bac感受态细胞。结果显示，ORF3、ORF5基因长度分别为765bp、603bp，发现核苷酸的同源性分别为88.0％—99.2％、87.3％—99.0％，获得了重组转座子rBacmid-ORF3、rBacmid-ORF5。Two pairs of primers for ORF3 and ORF5 genes of porcine reproductive and respiratory syn- drome virus （PRRSV） were designed and synthesized according to the published genome sequences of PRRSV（CH-1a,AY032626）available in GenBank. ORF3 and ORF5 genes of PRRSV Lanzhou strain were amplified by RT-PCR,sequenced and compared with other 13 strains of PRRSV. The amplified ORF3 and ORF5 genes were cloned respectively into pFastBacHTA vector to construct recombinant plasmids pFast-BacHTA-ORF3 and pFastBacHTA-ORF5. The positive pFastBacHTA-ORF3 and pFastBacHTA-ORF5 identified by enzymatic digestion and sequencing were transformed into DH10Bac competent cells, respec-tively. Sequence analysis showed that ORF3 and ORF5 genes consisted of 765 bp and 603 bp,respectively. The homologies of ORF3 and ORF5 genes were 88.0%-99.2% and 87.3%-99.0% in nueleotide sequence among Lanzhou strain and the reported 13 strains, respectively. Two recombinant shuttle vehicles rBacmid-ORF3 and rBacmid-ORF5 were obtained.

关 键 词：猪生殖与呼吸综合征病毒 ORF3基因 ORF5基因 克隆 杆状病毒表达载体 

分 类 号：S852.659[农业科学—基础兽医学] Q786[农业科学—兽医学]