马传染性贫血病毒基因非编码区LTR嵌合克隆的构建  

Construction of a Chimeric Infectious Clone of Chinese Equine Infectious Anemia Virus by Partial LTR Substitution

作　　者：魏丽丽[1] 王晓钧[1] 王盈[2] 粱华[3] 沈韬[3] 李景鹏[2] 张晓燕[3] 相文华[1] 邵一鸣 沈荣显[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150001 [2]东北农业大学生命科学院,黑龙江哈尔滨150030 [3]中国疾病预防控制中心性病艾滋病预防控制中心,北京100050

出　　处：《中国病毒学》2005年第2期149-154,共6页Virologica Sinica

基　　金：国家自然科学基金青年基金资助(30200200);科技部基础科研前期重大专项

摘　　要：在已有全长感染性克隆pLGFD3 8 和pD70344 的基础上,根据马传贫弱毒疫苗致弱过程中不同代次毒株LTR序列的分析,在LTR U3区选取特定的酶切位点对EIAV非编码区LTR基因进行了部分替换。将替换的全基因克隆转染驴胎皮肤细胞(FDD)并以驴白细胞(DL)传代,用逆转录酶活性检测、RT PCR方法及Real time RT PCR验证其感染性。结果发现,其衍生病毒感染上述两种细胞均出现明显的细胞病变;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EIAV颗粒。pLGFD9 12 嵌合克隆衍生病毒与其父本克隆衍生病毒pLGFD3 8具有相似的复制水平,pLGFD9 12嵌合克隆衍生病毒在DL细胞上复制水平略高于FDD细胞。此结果为进一步深入研究LTR对马传染性贫血病毒复制水平和毒力的影响奠定了基础。According to mutations occurred in the passages during the Equine infectious anemia virus (EIAV) vaccine attenuation, a full length chimeric gene clone, pLGFD9-12, was constructed successfully at a backbone of clone pLGFD3-8 by substitution with LTR U3 region of a virulent EIAV strain. The pLGFD9-12 was used to transfect fetal donkey dermal (FDD) cells and passaged in donkey leukocyte (DL) culture. The cell cultures were monitored by RT-PCR, reverse transcriptase activity assay and real-time RT-PCR. The results of RT activity and RT-PCR were positive in the supernatant of cell cultures and viruses particles were also clearly observed under electron microscope. The replication of pLGFD9-12 chimeric viruses and its parental virus pLGFD3-8 has no obvious differences. The level of replication of the pLGFD9-12 chimeric clone cultured in DL cells was higher than that in FDD cells. The characteristics on virulence and replication ability of pLGFD9-12 will be evaluated in vivo.

关 键 词：马传染性贫血病毒 基因替换 嵌合病毒 LTR 

分 类 号：S852.65[农业科学—基础兽医学]