马传染性贫血病毒N-连接糖基化回复突变的感染性克隆构建

Construction of an Infectious Clone of Equine Infectious Anemia Virus by N-glycosylation Revers-mutations

摘要:

根据马传贫强毒株EIAV-L和疫苗株EIAV-FDD表面蛋白gp90的N-连接糖基化的变化规律,采用PCR定点突变的方法, 对全长感染性克隆pLGFD3-8上的N-连接糖基化的差异区域进行改造后, 构建成含有3个N-连接糖基化位点突变的感染性克隆pLGN191N236N246。将其转染驴胎皮肤细胞(FDD),通过用逆转录酶活性、间接免疫荧光和RT-PCR方法检测而确定其感染性。结果表明, 在FDD细胞中盲传三代后, 在细胞培养物中可检测到逆转录酶活性, RT-PCR和间接免疫荧光检测均呈阳性, 电镜下见到典型的EIAV颗粒。这一结果可能对N-连接糖基化在我国马传贫弱毒疫苗致弱机理的作用研究而奠定良好的基础。

关键词:马传染性贫血病毒; N-连接糖基化; 定点突变; 感染性克隆

Abstract:

To elucidate the role of N-glycosylation in fetal donkey dermal cell (FDD)-attenuated equine infectious anemia virus (EIAV), we constructed an N-glycosylation reverse-mutation molecular clone, pLGN191N236N246. This viral molecular clone was derived from the infectious clone pLGFD3-8 by site-directed mutagenesis. This clone was used to transfect fetal donkey dermal (FDD) cells. Infectious characteristics of transfectants were monitored by RT-PCR, indirect immune fluorescence and reverse transcriptase activity assay. After three passages in FDD cells, viral replications in the supernatant of cell cultures were detected by all the above three methods and viral particles were clearly observed by electron microscopy. The construction of the N-glycosylation reverse-mutation infectious clone provides a solid basis for further study of the mechanism of attenuated pathogenesis and in-creased immune protection of EIAV attenuated vaccines.

Key words:equine infectious anemia virus (EIAV); N-glycosylation; site-directed mutagenesis; infectious clone