猪瘟病毒低温诱变疫苗Thiverval株感染性克隆的构建及病毒拯救  

Recovery of the Infectious Classical Swine Fever Virus from the Cloned cDNA of Low Temperature Attenuated Thiverval Strain

作　　者：范运峰[1,2] 赵启祖[1] 赵耘[1] 邹兴启[1] 徐璐[1] 张仲秋[1,2] 王琴[1] 宁宜宝[1] 

机构地区：[1]中国兽医药品监察所,北京100081 [2]中国动物疫病预防控制中心,北京100026

出　　处：《畜牧兽医学报》2009年第9期1420-1425,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(30571377);国家863计划(2006AA10A204)

摘　　要：采用高保真DNA聚合酶,分7个片段扩增猪瘟病毒(CSFV)Thiverval株全基因组序列,克隆至pMD18-T载体并测序。经适当的连接策略将各片段连接克隆至低拷贝载体质粒pAC/F101,同时对病毒基因组5′和3′末端进行修饰,构建出含有T7启动子、榔头状(HH)核酶基因、CSFV Thiverval全基因组、丁型肝炎病毒(HDV)核酶基因和T7终止子的重组质粒pAC/F101/T1-7。利用T7 RNA聚合酶将线性化重组质粒pAC/F101/T1-7体外转录成基因组RNA,再使用脂质体转染将体外转录RNA转染PK-15细胞。通过传代、RT-PCR、免疫过氧化物酶细胞单层试验鉴定,表明成功的从感染性克隆拯救出活病毒粒子。猪瘟病毒低温诱变疫苗"Thiverval"株感染性克隆的构建及病毒的成功拯救,为进一步研究CSFV疫苗株的致弱机制提供了重要工具。Seven eDNA fragments covered the whole genome of classical swine fever virus （CSFV） Thiverval strain were amplified by reverse transcription PCR with PfuUltraTM High-Fidelity DNA Polymerase. Products were ligated into pMD18-T vector and sequenced. Using the appropriate ligation strategy, the whole genome eDNA were constructed into a low-copy pAC/F101 vector. The recombinant plasmid was designated as pAC/F101/T1-7, which contained the complete genome of CSFV Thiverval strain with T7 promoter, hammerhead ribozyme and HDV ribozyme, T7 termination sequence at both sides. The viral RNA was synthesized in vitro with T7 RNA polymerase, and transfected into PK-15 cells with lipofectamine 2000. After the cell passage, RT-PCR detection and immunoperoxidase monolayer assay with E2 monoclonal antibody, the results indicated that the virus had been rescued from the Thiverval strain full-length eDNA clone. Successful construction of infectious clone of CSFV Thiverval strain provided a useful tool for further study the attenuated mechanism of CSFV vaccine strains.

关 键 词：猪瘟病毒 疫苗Thiverval株 感染性克隆 病毒拯救 

分 类 号：S852.651[农业科学—基础兽医学]