马传染性贫血病毒囊膜基因gp90 V4区糖基化回复突变感染性克隆的构建  

Construction of an infectious clone within the V4 region of gp90 surface protein of EIAV by N-glycosylation revers-mutations

作　　者：韩秀娥[1,2] 张萍[3] 王雪峰[1] 孔宪刚[1] 周建华[1] 相文华[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/大动物病研究室,黑龙江哈尔滨150001 [2]东北农业大学黑龙江省乳品工业技术开发中心,黑龙江哈尔滨150001 [3]东北农业大学动物医学学院,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2009年第3期165-169,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家"973项目"(2001CCA00600);黑龙江省发展高新技术产业专项资金项目(FW05B007)

摘　　要：为了阐明我国马传染性贫血病毒(EIAV)弱毒疫苗致弱及免疫保护的分子机制,本研究分析了疫苗株及其亲本强毒株共34个囊膜基因序列。结果发现疫苗株在膜基因gp90V4区潜在的N-连接糖基化位点出现了稳定的碱基替换,使该位点消失。为研究囊膜糖基化的作用,以疫苗株全长感染性克隆pLGFD3-8为亲本,利用反向遗传技术对该突变位点进行了糖基化序列回复突变操作,构建了全基因感染性克隆pLGFDg9。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3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。The Chinese equine infectious anemia virus （EIAV） donkey-leukocyte attenuated vaccine （DLV） provides a unique model system for study of the attenuation mechanism and the immunological control of lentivirus replication. In this study, we compared gp90 sequences of the env gene from both virulent and attenuated EIAV strains and found that all attenuated strains lost the potential N-linked glycosylation sites in the V4 region. To determine the action of this mutation, we constructed the infectious clone pLGFDg9 to restore the potential N-linked glycosylation site. This clone was transfected into fetal donkey dermal （FDD） cells and virus replication was monitored by RT-PCR, indirect immune fluorescence and reverse transcriptase activity assay. The results showed that, after 3 passages in FDD cells, virus replication in the cell cultures supernatant was detected by all three methods and viral particles were clearly observed by electron microscopy. The N-glycosylation reverse-mutation infectious clone provides a solid base for further study of the mechanism of attenuation and increased immune protection of EIAV attenuated vaccines.

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 

分 类 号：S852.659.3[农业科学—基础兽医学]