马传染性贫血病毒弱毒疫苗株感染性分子克隆的构建  

Infectious Molecular Clone Derived from Equine Infectious Anemia Virus Chinese Donkey Leukocyte Attenuated Strain

作　　者：王柳[1] 童光志[1] 仇华吉[1] 谷守林[1] 涂亚斌[1] 杨志彪[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所

出　　处：《中国农业科学》2003年第12期1560-1565,共6页Scientia Agricultura Sinica

基　　金：国家 8 6 3计划 ( 2 0 0 1AA2 2 30 4 1 );国家自然科学基金 ( 30 1 70 70 6 );中国博士后科学基金资助项目

摘　　要：采用PCR方法分 3段扩增出马传染性贫血病毒驴白细胞弱毒疫苗株 (EIAVDLA)的前病毒DNA ,这 3个片段覆盖马传染性贫血病毒的全部基因组 ,PCR产物经克隆后顺次连接 ,获得 1个含有EIAV全基因 (8.0kb)的重组质粒 ,将其命名为p8.0。将此 8.0kbEIAV全基因再亚克隆到含有一完整EIAVDLA株长末端重复序列的质粒中 ,获得一含有EIAV驴白细胞弱毒前病毒全基因的重组质粒 ,将其命名为p8.2 ,经核苷酸序列分析 ,证明p8.2含有EIAV前病毒的全基因。用p8.2转染驴白细胞 ,将其作为种毒进行传代 ,于感染该克隆毒的细胞培养上清中检测出了反转录酶活性 ,说明在驴白细胞中由p8.2衍生出了EIA病毒。驴白细胞经该克隆毒感染后 ,第 4天出现病变 ,经透射电镜可观察到典型的马传染性贫血病毒粒子 ,进一步证明p8.2具有感染性 ,笔者获得了马传染性贫血病毒驴白细胞弱毒疫苗株的感染性分子克隆 。In this study, the proviral DNA was extracted from do nk ey leukocyte infected with Chinese donkey leukocyte attenuated (DLA) strain of e quine infectious anemia virus (EIAV). Three fragments covering the entire EIAV g enome were amplified by PCR using the proviral DNA as template. A recombinant pl asmid (designated as p8.0) was obtaining by ligating these three fragments. The p8.0 was subcloned into a plasmid (designated as p8.2) containing entire LTR of EIAV DLA. The complete nucleotide sequence of DLA strain of EIAV was determined by sequencing the p 8.2 . The purified p8.2 was used to transfect donkey leuko cyte cultures. The cell culture supernatants were tested to be positive for reve rse transcriptase activity. Cytopathogenic effects were observed by 4 days post -infection in donkey leukocyte infected with the p8.2-derived virus. EIAV-lik e virions wer e also observed by electronic microscope in donkey leukocyte infected with the p 8.2-derived virus. The result indicated that p8.2 was an infectious molecular c lone of DLA strain of EIAV. The infectious molecular clone will provide an impor tant tool for further study of mechanism of replication and attenuation of the E IAV DLA strain.

关 键 词：马 传染性贫血病毒 弱毒疫苗株 感染性 分子克隆 基因构建 

分 类 号：S852.52[农业科学—基础兽医学]