狂犬病街毒是一种嗜神经性，能引起几乎所有哺乳动物发生脑神经炎和100%致死性的病毒。其属于弹状病毒科，狂犬病病毒属，基因组为单股负链RNA病毒。基因组长约12kb，负责编码5种结构蛋白：核蛋白、磷蛋白、基质蛋白、糖蛋白和大转录蛋白。核蛋白、磷蛋白、大转录蛋白和病毒的基因组构成核蛋白体复合物（RNP），仅有它才能启动病毒复制和蛋白表达。 我国是狂犬病发病最为严重的地区之一，近些年发病人数逐年增加，每年的发病人数都在3000～4000之间。数据显示，人狂犬病例中93%都是由犬咬伤发病的，因此，犬的免疫预防成为对于预防人的狂犬病是非常重要的。 为获得活的病毒载体疫苗和便利的病毒检测方法。选用了有很好免疫原性，在我国广泛用于制备人狂犬病灭火苗、制备动物弱毒苗和灭活苗的Flury-LEP疫苗株，用于建立反向遗传学系统。在此基础上，表达绿色荧光蛋白的重组LEP-EGFP病毒被拯救，并进行了分析。本研究为研制以Flury-LEP株病毒活载体多价疫苗、高效安全的基因修饰疫苗奠定了基础，同时为检测病毒中和抗体提供了更为方便和经济新方法。 1. 为获得狂犬病病毒载体，在建立Flury-LEP疫苗株反向遗传学基础之上，通过已知序列设计引物，经SOE-PCR在Flury-LEP cDNA 4096nt处突变出Pme I酶切位点。选用EGFP为报告基因，利用PCR引物设计技术在此ORF两端引入本疫苗株L蛋白转录开始和终止序列，以及Pme I位点，将此报告基因克隆入病毒cDNA Pme I位点处构建了表达绿色荧光蛋白（GFP）的重组Flury-LEP基因组cDNA克隆pCI-LEP-EGFP。将PCI-LEP-EGFP和辅助质粒PCAGG-N、PCAGG-P、PCAGG-L共转染293T细胞，荧光显示成功拯救重组病毒LEP-EGFP。 2. 为获得狂犬病病毒LEP-EGFP作为载体的生物学特性。LEP-EGFP和野生型LEP（wtLEP）以M.O.I.为0.01分别感染NA细胞和BHK-21细胞，在24h、72h和120h取样滴定，生长动力学特性与亲本株无明显差异。wtLEP和LEP-EGFP分别颅内接种Balb/c雌性小鼠，Reed-Muench法计算LD50分别为6.3个FFU和8.8个FFU，两毒株小鼠的致病性没有显着差异。 LEP-EGFP在BHK-21细胞中连续传代9次，各代保持EGFP的稳定表达及生物学特性不变。 3. 为鉴定EGFP表达是否影响Flury-LEP活疫苗载体免疫原性，和开发更为方便、快捷中和抗体检测方法。wtLEP和LEP-EGFP分别以 股内侧免疫7周龄Balb/c，三周后采血，所获得的抗体滴度显示了很好的一致性（P＞0.05）。比格犬免疫Flury-LEP弱毒苗或灭活苗三周后获得血清，经IFAT (Indirect fluorescent antibody test)和LEP-EGFP直接荧光下检测中和50%攻击毒株抗体滴度，两种方法检测的中和抗体滴度显示很好的相似性（P＞0.05）。 本研究获得了重组LEP-EGFP疫苗载体，其在致病性、免疫原性和生长动力学与其母本相似。其用于中和抗体检测，结果与IFAT方法有很好的相似性。本研究将为狂犬病病毒载体活疫苗和中和抗体检测发挥重要作用。

Rabies street virus targets the central nervous system (CNS) and causes fatal encephalitis in almost all species of mammals, the case fatality ratio (CFR) is 100 percent approximately. Rabies virus belongs to the genus Lyssavirus of the family Rhabdoviridae and has an unsegmented negative-sense RNA as the viral genome. The genome is about 12 kb in length and encodes five genes for the structural proteins: NP, PP, MP, GP, and LP. NP, PP, LP and the viral genomic RNA compose a ribonucleoprotein complex (RNP), only this one could initiate viral gene replication and expression. China is one of the most severe regions of rabies, and the number of rabies cases is increasing consecutively during recent years. The dead cases are about 3000-4000 every year in china in last several years.The data show that the 93% of human rabies cases was caused by canine bite injure, so canine immunized rabies vaccine is most important to prevent from human rabies. To prepare live viral vector vaccines of rabies virus and convenient method for virus neutralization antibodies (VNA) detecting, based on the establishment of reverse genetic operation system of rabies virus Flury-LEP strain, this strain is widely used as inactivated vaccine of human rabies, and live-attenuated vaccine and inactivated one of animals in China, and its immunogenicity is very good. A recombinant rabies virus LEP-EGFP strain expressing enhanced green fluorescent protein (EGFP) was generated and analyzed. Our research on recombinant virus LEP-EGFP provided a useful platform for development of novel polyvalent vaccine and gene modified vaccine, and convenient, economical and reliable method for virus neutralization antibodies (VNA) detecting. 1. To prepare rabies virus vector, based on establishment of reverse genetic system of rabies virus Flury-LEP strain, Flury-LEP cDNA with 4096nt Pme I mutant was constructed by SOE-PCR, and the primers was devised according to acquired sequence. EGFP ORF as a reporter gene was modified with start point and end point of large transcription protein of rabies virus, and this one was inserted in Pme I of a full length cDNA clone, named PCI-LEP-eGFP. 293T Cells were transfected with PCI-LEP-EGFP, PCAGG-N, PCAGG-P, PCAGG-L, the result showed that LEP-EGFP was successfully rescued. 2. This study aimed to obtain the biological characteristics of LEP-EGFP vector. NA cells and BHK-21 cells were infected with the wild type LEP (wtLEP) or LEP-EGFP virus at MOI = 0.01. At 24, 72 and 120h after the infection, supernatants of the culture fluid were subjected with virus titration assay, the replication kinetics of LEP-EGFP did not differ significantly from one of wtLEP. Bal/BC mice were infected by intracerebral inoculation with wtLEP and LEP-EGFP, LEP-EGFP LD50=6.3 FFU is similar in pathogenicity to wtLEP LD50=8.8 FFU. The EGFP expression of LEP-EGFP was stable for at least nine passages in BHK-21 cells. 3. To obtain whether the immunogenicity of live viral vector of LEP-EGFP was influenced by EGFP expression, convenient method for virus neutralization antibodies (VNA) detecting was prepared. Seven-week-old Bal/BC was immunized with 105FFU wtLEP and LEP-EGFP at medial vastus muscle, and the antibody titer of serum after three weeks showed good agreement (P＞0.05). The LEP-EGFP strain could be used as challenge virus in the 50% virus neutralization assay of canine serum from beagles immunized Flury-LEP live-attenuated vaccine and inactivated one after three weeks , the results obtained by the both LEP-EGFP method and IFAT showed good agreement （P＞0.05）. Taking above-mentioned data, this study can be concluded that LEP-EGFP is similar in pathogenicity, the replication kinetics and immunogenicity to that of the wild type. The EGFP expression of LEP-EGFP was stable for at least nine passages. The result between LEP-EGFP of VNA assay and IFAT showed good agreement. It might play a significant role in developing recoment rabies vaccine and VNA assay.