摘要
提取山羊痘病毒温度敏感株基因组DNA，用HindⅢ酶切，随机克隆到pUC18质粒中，限制性内切酶片段电泳分析和序列测定结果表明，克隆到4个不同大小的HindⅢ片段pUA、pUB、pUC和pUD。随后，将它们和羊痘病毒Pellor（AY077835）株进行了同源性比较，发现核苷酸同源性为90。0％～94．5％，氨基酸同源性为83．0％～91．4％。选取克隆或亚克隆后的单一酶切位点，插入P11P7.5-LacZ报告基因，构建了pUAl、pUBl、pUCl和pUDl共4个重组载体质粒。通过脂质体分别转染山羊痘病毒感染的BHK-21细胞，在X-gal存在条件下经蓝白斑筛选重组病毒，获得1株重组病毒，证明筛选出了1个复制非必需区片段。
Genomic DNA was rified by sucrose density gradient extracted from the goatpox virus（GPV） temperature-sensitive strain pucentrifugation. Four fragments digested by Hind Ⅲ from the GPV DNA were randomly cloned into pUC18 vector and the clones pUA,pUB,pUC and pUD with the inserted DNA of different sizes were obtained. Compared with the Pellor（AY077835）strain, the similarity of nucleotide sequence and amino acids sequence were 90.0%-94.5% and 83.0%-91.4% ,respectively. The P11 P7.5-LacZ report gene was inserted in the unique sites of pUA,pUB,pUC and pUD,respectively. The acquired plasmids pUA1, pUB1, pUC1 and pUD1 were transfected with GPV into BHK-21, respectively. After plaque selection, passages and purifications with X-gal staining,one cloned GPV fragment was identified to be non-essential regions for the virus replication.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2007年第9期737-741,共5页
Chinese Veterinary Science
基金
国家科技基础平台项目(2005DKA21205-3)
关键词
山羊痘病毒
非必需区
基因克隆
序列分析
转染
goatpox virus
non-essential region（NER） gene cloning
sequence analysis
transfection
