SHEN Tao, ZHANG Xiao-yan, TONG Xiao, FAN Xiu-juan, LIANG Hua, MA Yan, XIANG Wen-hua, SHEN Xian-rong and SHAO Yi-ming. Construction and In Vitro Evaluation of EIAV Full-length Infectious Clones with Site-directed Retromutations[J]. Virologica Sinica, 2005, 20(1): 55-60.
Citation: SHEN Tao, ZHANG Xiao-yan, TONG Xiao, FAN Xiu-juan, LIANG Hua, MA Yan, XIANG Wen-hua, SHEN Xian-rong, SHAO Yi-ming. Construction and In Vitro Evaluation of EIAV Full-length Infectious Clones with Site-directed Retromutations .VIROLOGICA SINICA, 2005, 20(1) : 55-60.

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

  • 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。

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

  • Based on t he infectious clone p FD328 wit hin EIAV f ull2lengt h 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 site2 directed mutagenesis , were constructed successfully. These clones were used to t ransfect fetal donkey dermal (FDD) cell s and donkey differentiated monocyte macrop hages (DL ) , and their infectious characteristics were monitored by RT-PCR and rever set ranscriptase activity assay. The results indicated that after five generations passaged in FDD cell s t hen five generations in differentiated monocyte2macrop hages , RT activity and RT2PCR were found to be obviously positive in cell cult ure supernatant and viruses particles were al so clearly observed under electron microscope. Nevertheless , t he final viral titer s 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 t heir parental virus pL GFD328 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.

  • 加载中
  • 加载中

Article Metrics

Article views(4131) PDF downloads(998) Cited by(0)

Related
Proportional views
    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

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

    • 1. 

    Abstract: Based on t he infectious clone p FD328 wit hin EIAV f ull2lengt h 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 site2 directed mutagenesis , were constructed successfully. These clones were used to t ransfect fetal donkey dermal (FDD) cell s and donkey differentiated monocyte macrop hages (DL ) , and their infectious characteristics were monitored by RT-PCR and rever set ranscriptase activity assay. The results indicated that after five generations passaged in FDD cell s t hen five generations in differentiated monocyte2macrop hages , RT activity and RT2PCR were found to be obviously positive in cell cult ure supernatant and viruses particles were al so clearly observed under electron microscope. Nevertheless , t he final viral titer s 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 t heir parental virus pL GFD328 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.

    Relative (20)

    目录

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return