马传染性贫血病毒弱毒株LTR点突变型嵌合感染性克隆的构建  

Construction of a chimeric clone of Chinese equine infectious anemia virus by site-mutation in the LTR region

作　　者：左咏梅[1] 吕晓玲[1] 赵立平[1] 李庆章[2] 郝艳红[2] 沈荣显[1] 相文华[1] 王晓钧[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/大动物传染病研究室,黑龙江哈尔滨150001 [2]东北农业大学,黑龙江哈尔滨150030

出　　处：《中国预防兽医学报》2007年第8期569-574,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金青年基金项目(30200200)

摘　　要：马传染性贫血病毒(Equine infectious anemia virus,EIAV)长末端重复序列(Long terminal repeat,LTR)是基因组中高度变异区之一,EIAV LTR的变异对于指导病毒的复制和病毒致病性具有重要的生物学意义。为了阐明我国马传染性贫血病毒弱毒疫苗毒力致弱的分子机制,由马传贫强毒至弱毒致弱过程中不同代次毒株LTR序列的分析,以驴胎皮肤弱毒株疫苗全长感染性克隆pLGFD3-8为父本,选取LTR R区的TAR起始碱基,poly(A)附加位点,采用反向遗传操作对其位点进行PCR体外定点突变,将弱毒序列构建的逆向点突变型全基因克隆转染到驴胎皮肤细胞(FDD)并在FDD上传代,通过逆转录酶活性(RT)检测、RT-PCR方法及real-time RT PCR检测并验证其感染性。检测结果为构建的逆向点突变型感染性克隆在FDD上被拯救,其衍生病毒感染的FDD上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pLGFD-M点突变型嵌合克隆衍生病毒与其父本克隆衍生病毒pLGFD3-8复制水平相似。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。Equine infectious anemia virus （EIAV） long terminal repeat sequence （LTR） is one of the highly variant regions of the virus genome and has a biological significance in virus replication and pathogenicity. In order to investigate the molecular mechanism of the virus attenuation, the Chinese EIAV attenuated vaccine was developed by passaging a highly virulent EIAV strain in donkey leucocytes culture in vitro for more than 110 times until completely lost pathogenicity. A full length chimeric clone with a mutated LTR, designated pLGFD-M, was constructed based on infectious clone pLGFD3-8 by PCR directed site mutations in LTR TAR and the poly （A） sequences, pLGFD-M was used to transfect the fetal donkey dermal （FDD） cells and the cell culture were monitored by RT-PCR, reverse transcriptase activity （RT） assay and real-time RT-PCR. The results showed that the chimeric viruses were successfully rescued from the clone. Both RT activity and RT-PCR were positive in the supernatant of cell cultures and typical CPE was developed. Virus particles were clearly observed under electron microscope. The replicationdynamics of pLGFD-M chimeric viruses was similar to that of the parental virus pLGFD3-8. These results laid foundation for further study of molecular mechanisms of LTR effects on the replication and attenuation of DLA-EIAV.

关 键 词：马传染性贫血病毒 定点突变 嵌合病毒 

分 类 号：Q784[生物学—分子生物学] S852.65[农业科学—基础兽医学]