马传染性贫血病病毒弱毒疫苗株感染性分子克隆的构建

Characterization of Infectious Molecular Clone of Equine Infectious Anemia Virus Chinese Donkey Leukocyte Attenuated Strain

采用ＰＣＲ方法分三段扩增出马传染性贫血病毒驴白细胞弱毒疫苗株（ＥＬＡＶ ＤＬＡ）的前病毒ＤＮＡ，这三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，获得一个含有ＥＬＡＶ全基因（８．０Ｋｂ）的重组质粒，将其命名为ｐ８．０。将此８．０Ｋｂ ＥＩＡＶ全基因再亚克隆到含有一完整 ＥＩＡＶ ＤＬＡ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，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，我们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，为进一步在分子水平上阐明我国ＥＩＡＶ疫苗株的减毒机理和免疫保护机制奠定了基础。

In this study, proviral DNA was extracted from donkey leukocyte infected by Chinese donkey leukocyte attenuated (DLA) strain of equine infectious anemia virus(EIAV). Three fragments covering the entire EIAV genome were amplified by PCR using the proviral DNA as template. These PCR products were cloned. A recombinant plasmid (designated as p8 .0) containing entire EIAV genome was obtained by ligat- ing these three fragments. p8. 0 was subcloned into a plasmid containing a long terminal repeat sequen...