表达荧光素酶重组RABV rERA-Luc株的构建及其生物学特性的研究  

Generation of a recombinant rabies virus ERA strain expressing luciferase and its biological characteristics

作　　者：王娅荣 帅磊[2] 王金明 舒伦 金山[1] 步志高[2] WANG Ya-rong;SHUAI Lei;WANG Jin-ming;SHU Lun;JIN Shan;BU Zhi-gao(College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot 010018,China;State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;College of Animal Science,Inner Mongolia Agricultural University,Hohhot 010018,China)

机构地区：[1]内蒙古农业大学兽医学院农业部动物临床诊疗技术重点实验室,内蒙古呼和浩特010018 [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,黑龙江哈尔滨150069 [3]内蒙古农业大学动物科学学院,内蒙古呼和浩特010018

出　　处：《中国预防兽医学报》2021年第6期577-583,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金青年科学基金(31800138)。

摘　　要：为建立一种能够实现病毒活体示踪的狂犬病病毒(RABV),以有效地研究RABV的侵染机制,本研究基于重组RABV rERA载体,利用反向遗传技术构建了包含rERA全长基因组cDNA及萤火虫荧光素酶(Luc)基因的重组质粒,并经Pme I酶切鉴定正确后,将该质粒与辅助质粒共转染鼠肾成纤维细胞(BSR),4 d后收集上清液,在BSR细胞盲传3代后收集上清液,经RT-PCR扩增结果显示,获得了Luc基因的目的条带;间接免疫荧光试验(IFA)鉴定结果显示,上清液接种的细胞出现绿色荧光;将上清液中的重组病毒纯化后电镜观察结果显示重组病毒与亲本病毒均呈弹头状,Western blot结果显示纯化后重组病毒感染BSR细胞后均能够表达RABV 5种结构蛋白,与亲本病毒蛋白表达情况一致,上述结果表明获得了与亲本病毒性状一致的重组病毒,命名为rERALuc。将重组病毒在BSR细胞中连续传15代,每代均经PT-PCR及测序鉴定,并经IFA测定每代重组病毒的病毒滴度,分析重组病毒增殖稳定性;测定各代次重组病毒感染BSR细胞48 h后的荧光素酶活性,分析其遗传稳定性。各代次重组病毒的RT-PCR及测序结果显示,Luc基因均未发生突变;IFA结果显示,F5代及以后代次的重组病毒与亲本病毒在体外的病毒滴度无显著差异并能稳定传至15代;荧光素酶活性测定结果显示,重组病毒荧光素酶活性随传代次数的增加逐渐升高且从第5代以后的重组病毒均能够稳定表达荧光素酶,而亲本病毒荧光素酶活性一直维持较低水平,表明重组病毒具有较强的增殖及遗传稳定性。将重组病毒与亲本病毒分别感染BSR细胞和小鼠神经瘤细胞(NA),感染后不同时间(24 h、48 h、72 h、96 h、120 h、144 h),分别经IFA测定两种病毒的病毒滴度,绘制生长曲线,并测定二者的荧光素酶活性。结果显示,重组病毒与亲本病毒在这两种细胞中的生长特性均无显著差异;且rERA-Luc的荧光素酶活性均随感In order to trace rabies virus(RABV) in vivo, we constructed a recombinant RABV ERA virus expressing firefly luciferase(Luc) gene using reverse genetics technology. The recombinant virus was named rERA-Luc. RT-PCR confirmed the presence of Luc in the rescued virus genome;indirect immunofluorescence assay(IFA) showed the recombinant virus reacted with anti-RABV serum. Electron microscopy showed the recombinant virus maintained the bullet-shaped morphology as compared to the ERA virus. Western blot results showed the presence of RABV structural proteins in the purified rERA-Luc. Serial passage(15 passages in BSR cells) in cells showed rERA-Lucis genetically stable and capable of expressing luciferase. rERA-Luc also showed the similar growth kinetics to that of the ERA virus in NA and BSR cells. Compared to the ERA virus, 104 ffu rERA-Luc infected mice either by intramuscular or intracranial injection demonstrated similar clinical symptoms, body weight change and survival rate.Thus, the successful generation of rERA-Luc provides a powerful tool to study RABV in vivo distribution and pathogenesis.

关 键 词：狂犬病病毒 荧光素酶 生物学特性 

分 类 号：S852.65[农业科学—基础兽医学]