Construction and Characterization of EIAV Vaccine Infectious Clones Labeled with FLAGTM or  6 His-tag

SHEN Tao; ZHANG Xiao-yan; NAN Chang-long; TONG Xiao; FAN Xiu-juan; SHEN Rong-xian; SHAO Yi-ming

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June 2005

SHEN Tao, ZHANG Xiao-yan, NAN Chang-long, TONG Xiao, FAN Xiu-juan, SHEN Rong-xian and SHAO Yi-ming. Construction and Characterization of EIAV Vaccine Infectious Clones Labeled with FLAGTM or 6 His-tag[J]. Virologica Sinica, 2005, 20(3): 257-261.

Citation: SHEN Tao, ZHANG Xiao-yan, NAN Chang-long, TONG Xiao, FAN Xiu-juan, SHEN Rong-xian, SHAO Yi-ming. Construction and Characterization of EIAV Vaccine Infectious Clones Labeled with FLAGTM or 6 His-tag .VIROLOGICA SINICA, 2005, 20(3) : 257-261.

特异性标记EIAV感染性克隆的构建及鉴定

沈弢 ¹ ,
张晓燕 ^1* ,
南昌龙 ¹ ,
童骁 ^1,2 ,
范秀娟 ¹ ,
沈荣显 ³ ,
邵一鸣 ¹

1.
1.中国疾病预防控制中心性病艾滋病预防控制中心病毒免疫室哈尔滨 2.中国科学院武汉病毒所武汉 3.北京中国农业科学院哈尔滨兽医研究所

摘要

用FLAGTM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pFD3FLAG和pFD3HISADD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pFD3FLAGRT酶活性显示弱阳性,未见明显的细胞病变;pFD3HISADD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。
- 马传染性贫血病毒
- , 感染性克隆
- , S2基因
- , 细胞病变效应
- , 分子标签

Construction and Characterization of EIAV Vaccine Infectious Clones Labeled with FLAGTM or 6 His-tag

Abstract

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.
- Equine infectious anemia virus
- , Infectious clone
- , S2 gene
- , Cytopathic effect
- , Molecular tag

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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

Construction and Characterization of EIAV Vaccine Infectious Clones Labeled with FLAGTM or 6 His-tag

Abstract: 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.