中文摘要：

为避免细胞外转录步骤,使RHDV感染性克隆的转录和病毒拯救过程一步化,试验通过PCR反应在RHDV cDNA序列的5′端添加T7启动子核心序列,3′端添加具有自身切割功能的核酶核心序列.然后,将RHDV基因组的不同cDNA片段装配到pTVT转录载体中,构建了含有RHDV全长cDNA序列的重组质粒pTVT-RHDV.在转染能稳定表达T7RNA聚合酶的BHK细胞后,将收获的细胞培养物感染MA104细胞.分别用逆转录PCR、间接免疫荧光检测以及一步生长曲线等方法对拯救病毒进行了鉴定.结果表明,试验成功拯救出了RHDV,优化了RHDV感染性克隆的构建过程.

英文摘要：

The new infectious clone for rabbit hemorrhagic disease virus（RHDV） was successfully constructed,which avoided extracellular transcription step.In this process,the core sequence of T7 promoter and ribozyme were added to the 5′ and 3′ end of RHDV cDNA sequence respectively.The full length cDNA of RHDV was assembled into pTVT plasmid（named pTVTRHDV）,then pTVT-RHDV was transfected into BSR（BHK21 expressing T7 RNA polymerase）.After that MA104 cells was inoculated by the recovered cell cultures,and the rescued virus was identified by RT-PCR and IFA.The results showed that RHDV had been rescued and its infectious clone was successfully optimized too.