表达绿色荧光蛋白重组新城疫病毒LaSota疫苗株的构建  

Rescue of a recombinant Newcastle disease virus expressing the green fluorescent protein

作　　者：葛金英[1] 温志远[2] 王永[2] 鲍恩东[1] 步志高[2] 

机构地区：[1]南京农业大学动物医学院,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

出　　处：《微生物学报》2006年第4期547-551,I0003,共6页Acta Microbiologica Sinica

基　　金：国家十五科技攻关项目(2004BA519A19;2005BA711A10);国家"973项目"(2005CB523200)~~

摘　　要：新城疫病毒是理想的新型活病毒疫苗载体，具有巨大的优势和应用前景。采用生产实践中广泛应用、免疫效果良好的NDV LaSota弱毒疫苗株，建立了反向遗传操作系统。在此基础上，进一步构建了表达绿色荧光蛋白（GFP）的重组NDV基因组cDNA克隆，成功救获了重组病毒rLaSota-EGFP，病毒F1代尿囊病毒液按1×10^4 EID50接种9—10日龄SPF鸡胚尿囊腔，接种后分别于24h、48h、72h及96h收获尿囊液，检测平均HA滴度分别为2^8、2^10.3、2^11.3和2^11，每mL尿囊液病毒量EID50分别为10^8.64、10^9.22、10^9.21和10^9.64，重组病毒与亲本株生长滴度在相近时间达到峰值，生长动力学特性与亲本株无明显差异。各代次重组病毒按1×10^6 EID50病毒量接种9—10日龄SPF鸡胚，96h内完全不致死鸡胚。救获重组病毒保持了LaSota弱毒疫苗亲本毒株对鸡胚良好的高滴度生长适应和低致病特性，并且鸡胚连续传9代次仍保持GFP的稳定表达及生物学特性不变。重组病毒rLaSota-EGFP的成功救获为开展新城疫病毒活载体疫苗研制提供了可行的技术平台。A recombinant Newcastle disease virus （NDV） expressing the green fluorescent protein （GFP） was generated by applying reverse genetics techniques. The GFP open reading frame flanked by NDV transcription start and stop sequences was inserted between the phosphoprotein （P） and matrix protein （M） in a full-length cDNA clone of NDV Lasota vaccine strain. This plasmid transcribing antigenome RNA was cotransfected with helper plasmids expressing viral nucleoprotein, phosphoprotein and large protein into cells stably expressing T7 RNA polymerase. The rescued virus was first propagated in 10-day-old embryonated eggs and the allantoic fluid was used to infect primary chicken embryo fibroblasts （CEF） cells. The appearance of GFP in live infected cells confirmed further the recovery of a recombinant NDV（rNDV-GFP）expressing this reporter gene. Nine successive passages in embryonated chicken eggs were performed. Allantoic fluid samples were then titrated by a microtiter plate HA test. HA positive allantoic fluid were used for further egg passages.All the allantoic fluid samples were titrated by end point dilutions and infected cells were examined for the presence of GFP expression. To analyze virus growth, 10-day-old embryonated SPF chicken eggs were inoculated with 1×10^4 EID50 rNDV or rNDV-GFP. At 24,48,72 and 96 h p.i. the allantoic fluid of inoculated eggs containing live embryos was harvested and clarified by centrifugation. Supernatants were used for titration of EID50 in 10-day-old embryonated SPF chicken eggs. rNDV and rNDV-GFP grew to similar titers（109EID50/mL） . In order to test the virulence of rNDV-GFP, infectious allantoic fluid of rNDV-GFP were inoculated into embryonated SPF chicken eggs at 1×10^6 EID50. No dead embryonated egg was found within 96 hours. The replication kinetics and pathogenicity in SPF embryonated eggs of rNDV-GFP did not differ significantly from that of the parent virus. LaSota is a widely used NDV live vaccine strain. The reverse genetic system established for

关 键 词：新城疫 绿色荧光蛋白 反向遗传操作 活载体疫苗 

分 类 号：S852.4[农业科学—基础兽医学]