特异性标记EIAV感染性克隆的构建及鉴定

用FLAG^TM或6His对EIAV疫苗株全基因感染性克隆pFD3的S2分子进行分子标记，以建立EIAV疫苗株与野毒株感染鉴别诊断的方法。标记产物pFD3-FLAG和pFD3-HISADD转染驴胎皮细胞(FDD)后收获衍生病毒并用逆转录酶活性检测和PCR方法确定其感染性。在FDD细胞上盲传至第五代后收获细胞培养上清再感染驴单核巨噬细胞(DL)，盲传三代后pFD3-FLAGRT酶活性显示弱阳性，未见明显的细胞病变；pFD3-HISADD为强阳性，且细胞病变效应明显，在电镜下可见明显的病毒颗粒。与父本克隆pFD3相比，在细胞水平上二者复制特性有明显的不同。证明在DL细胞上S2基因的完整性是病毒复制很重要的因素。

Construction and Characterization of EIAV Vaccine Infectious Clones Labeled with FLAG~(TM) or 6 His-tag

To provide convenient and effective tool for differentiating analysis between EIAV vaccine and pathogenic strain, molecular marker of FLAG+{TM} or 6+*His was inserted into S2 gene of EIAV(equine infectious anemia virus) vaccine infectious clone pFD3, The resulted chimeric clones pFD3-FLAG and pFD3-HISADD were used to transfect fatal donkey dermal (FDD) cells. After 5 generations of in vitro passages, the supernatant was collected to further infect donkey blood leukocyte (DL) for 3 generations of passage. The replicative characteristic of pFD3-HISADD was similar to its parental clone pFD3, RT activity assay was strong positive with significant cytopathtic effect, and the virus particles could be observed under electron microscope. However, pFD3-FLAG showed a lower replication activity, RT activity was weak positive and no significant cytopathtic effect was observed. These resuts implicated that S2 gene was an important factor for viral replication in DL cell.