兔出血症病毒感染性克隆的优化  

Optimiation of infectious clone for rabbit hemorrhagic disease virus

作　　者：杨宗伟[1,2] 张伟伟[2,3] 陈宗艳[2] 李传峰[2] 吴润[1] 刘光清[2,4] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院上海兽医研究所,上海200241 [3]浙江师范大学,浙江金华321004 [4]兽医生物技术国家重点实验室,黑龙江哈尔滨150001

出　　处：《甘肃农业大学学报》2011年第3期8-12,共5页Journal of Gansu Agricultural University

基　　金：国家自然科学基金(30800045;30870114);兽医生物技术国家重点实验室开放基金(SKLVBF201004)

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.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.

关 键 词：兔出血症病毒 病毒拯救 全长CDNA 

分 类 号：S852.65[农业科学—基础兽医学]