摘要
从实验感染猪水泡病病毒(SVDV)的乳鼠组织中提取RNA,利用长距离的RACE技术,扩增出覆盖SVDVHK′1/70株全基因组的2个忠实性的cDNA重叠片段(3′PCR片段和5′PCR片段),分别克隆进pGEM-T Easy载体。利用AatⅡ和BssHⅡ酶切含5′PCR片段的重组质粒,回收目的片段,定向克隆于含3′PCR片段的重组质粒,构建出了SVDV HK′1/70株全长cDNA重组质粒,然后进行序列测定。结果表明,HK′1/70株基因组全基因组序列长7 401 nt(poly A除外),其中5′NCR长743 nt,该毒株蛋白编码区的核苷酸序列为6 558 nt,编码一个长2 185个氨基酸的聚合蛋白,3′NCR长102 nt,其后是至少含有74个A碱基的poly A尾。在HK′1/70株全长cDNA序列的5′端引入了T7启动子序列,在poly A3′端引入了Psp1406Ⅰ识别序列。通过序列同源性和进化树分析,结果表明,HK′1/70属于第Ⅱ抗原遗传群,SVDV与CB5的遗传关系最近,且位于CB5遗传进化树的分支上。HK′1/70株全长cDNA的序列测定及构建,为拯救SVDV和在分子水平进一步深入研究SVDV打下坚实的基础。
By RACE, 2 overlapping cDNA fragments （3′PCR and 5′PCR fragments） covering the full genome of swine vesicular disease virus strain HK′1/70 were amplified from total RNA extracted from experimentally infected suckling mice. These fragments were cloned into pGEM-T Easy vector, respectively. 5′PCR fragment was digested by enzymes of AatⅡ and BssHⅡ , and the AatⅡ -BssHⅡ -digested 5′PCR fragment was obtained and cloned into the recombinant pGEM-T Easy vector containing 3′PCR fragment,the recombinant plasmid encoding full-length cDNA of SVDV HK′1/70 strain was then obtained and sequenced. The results showed that the complete genome of HK′ 1/70 was 7401 nucleotides （nts） long （excluding the poly （A） tract） which encodes a single polyprotein of 2185 amino acids, a 5′untranslating region （UTR） of 743 nts, a 3′UTR of 102 nts and a poly （A） tail at least 74 adenines. T7 promoter was added at the 5′end of the full-length cDNA and an additional Pspl406I restriction site was added at the 3′end of poly （A） tail. The nucleotide and amino acid sequences were compared and phylogenetic analysis was used to examine the evolutionary relationships. The results showed that HK′1/70 belonged to the second antigenic group. SVD virus was antigenically closely related to Coxsackie B5 virus, and located on the branches of CB5 evolutionary tree. Successful construction of full-length cDNA clone of SVDV HK′1/70 strain lays foundation for rescuing SVDV effectively and enables further research of SVDV on molecular level.
出处
《病毒学报》
CAS
CSCD
北大核心
2007年第1期51-56,共6页
Chinese Journal of Virology
基金
国家重点基础研究发展计划(973)项目(2005CB523201)
国家高科技研究计划(863)项目(2003AA241110)
