逆向突变型EIAV全长基因组感染性克隆的构建及体外感染性评价  

Construction and In Vitro Evaluation of EIAV Full-length Infectious Clones with Site-directed Retro-mutations

作　　者：沈弢[1] 张晓燕[1] 童骁[1] 范秀娟[1] 梁华[1] 马燕[1] 相文华[2] 沈荣显[2] 邵一鸣 

机构地区：[1]中国疾病预防控制中心性病艾滋病预防控制中心,北京100050 [2]中国农业科学院哈尔滨兽医研究所,哈尔滨150001

出　　处：《中国病毒学》2005年第1期55-60,共6页Virologica Sinica

基　　金：基础研究重大项目前期研究专项 (2001CCA0060);国家自然科学基金(30371319)

摘　　要：在已有全长基因组感染性克隆 pLGFD3 8的基础上,按照疫苗制作过程中 EIAV结构基因的变化规律,对其中gag基因进行定点逆向回复改造。并在gag突变的基础上增加env 突变位点。将所改造的突变克隆转染驴胎皮肤细胞(FDD)以及驴单核巨噬细胞(DL),并用逆转录酶活性检测和 RT PCR方法验证其感染性。结果发现,衍生病毒感染上述两种细胞均出现明显的细胞病变效应;细胞培养上清可检测到 RT酶活性和 RT PCR阳性。电镜下可见大量典型的病毒颗粒。然而单核巨噬细胞培养病毒感染滴度要明显高于驴胎皮肤细胞培养病毒滴度。驴胎皮肤细胞内嵌合克隆衍生病毒和父本克隆衍生病毒的复制动力学比较分析显示前者的复制比后者略有滞后。此结果为深入研究马传染性贫血病毒致病的分子机制和疫苗保护机理奠定了基础。Based on the infectious clone pFD3-8 within EIAV full-length genome, and according to the variation of structural genes during vaccine preparation, several chimeric infectious clones involved in gag and env genes, modified by overlap PCR site-directed mutagenesis, were constructed successfully. These clones were used to transfect fetal donkey dermal (FDD) cells and donkey differentiated monocyte-macrophages (DL), and their infectious characteristics were monitored by RT-PCR and reverse transcriptase activity assay. The results indicated that after five generations passaged in FDD cells then five generations in differentiated monocyte-macrophages, RT activity and RT-PCR were found to be obviously positive in cell culture supernatant and viruses particles were also clearly observed under electron microscope. Nevertheless, the final viral titers of these ‘artificial’ viruses in DL culture were obviously higher than that in FDD culture. Analysis of the replicative characteristics between these chimeric viruses and their parental virus pLGFD3-8 showed that the formers slightly delayed in replication compared with the latter. All this provides a solid basis for further study of the pathogenic mechanism and immune protection against Chinese equine infectious anemia virus.

关 键 词：马传染性贫血病毒 定点突变 感染性克隆 

分 类 号：S852.65[农业科学—基础兽医学]