表达绿色荧光蛋白重组狂犬病病毒Flury-LEP的构建及其用于中和抗体检测的研究  

Construction of a recombinant rabies virus expressing enhanced green fluorescent protein for the detection of virus neutralization antibodies

作　　者：华涛[1,2] 陶丽红[2] 葛金英[2] 王喜军[2] 沈向真[1] 步志高[2] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]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/农业部兽医公共卫生重点开发实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2010年第8期581-585,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家"973"计划(2005CB523200)

摘　　要：为确定狂犬病病毒(RV)Flury-LEP株外源基因的插入位点及其中和抗体快速检测方法,本研究在建立了RVFlury-LEP株反向遗传操作系统的基础上,构建了表达增强型绿色荧光蛋白(EGFP)的重组LEP基因组cDNA克隆pCI-LEP-EGFP,并成功拯救出重组病毒(rLEP-EGFP)。rLEP-EGFP的生物学特性和野生型LEP(wtLEP)在NA细胞和BHK-21细胞中的生长动力学及对小鼠的致病性没有显著差异。rLEP-EGFP在BHK-21细胞中连续传代9次,各代次重组病毒均稳定表达EGFP。重组表达的EGFP可用于中和抗体检测,rLEP-EGFP免疫犬血清经间接免疫荧光(IFA)或荧光下直接观察检测RV中和抗体滴度,两种检测结果显示较好的相似性(p>0.05)。本研究为研制以Flury-LEP株活载体多价疫苗奠定了基础,并为病毒中和抗体的检测提供了更为快捷的新方法。To construct the recombinant rabies virus and develop a convenient method for virus neutralization antibodies （VNA） detection, a recombinant rabies virus rLEP-EGFP expressing enhanced green fluorescent protein （EGFP） was rescued based on infectious clone of rabies virus Flury-LEP strain. The biology characters of rLEP-EGFP were similar to that of the wild type LEP strain in respect of the growth dynamics in both NA cells and BHK-21 cells, and the pathogenicity in BALB/c mice. The EGFP expression of rLEP-EGFP was stable for at least nine passages in BHK-21 cells. The rLEP-EGFP could be used for detection of neutralization antibody in canine serum, and the results were as same as that of indirect immunofluorescence assay （p0.05）. This LEP-EGFP system provided an useful platform for development of novel polyvalent vaccine, and a convenient and economical method for VNA detection.

关 键 词：狂犬病病毒 Flury-LEP株 EGFP 反向遗传操作 多价疫苗 病毒中和抗体 

分 类 号：S852.65[农业科学—基础兽医学]