表达EGFP重组猪繁殖与呼吸综合征病毒的构建与鉴定  

CONSTRUCTION AND CHARACTERIZATION OF RECOMBINANT PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS EXPRESSING EGFP

作　　者：唐娜[1] 杨慧 刘磊 张松林[1] 于雪 孙培姣 沈志强[1,2] 肖跃强 TANG Na;YANG Hui;LIU Lei;ZHANG Song-lin;YU Xue;SUN Pei-jiao;SHEN Zhi-qiang;XIAO Yue-qiang(Shandong Binzhou Animal Science&Veterinary Medicine Academy,Binzhou 256600,China;Shandong Lvdu Bio-Sciences and Technology Co.Ltd.,Binzhou 256600,China)

机构地区：[1]山东省滨州畜牧兽医研究院,滨州256600 [2]山东绿都生物科技有限公司,滨州256600

出　　处：《中国动物传染病学报》2020年第4期39-46,共8页Chinese Journal of Animal Infectious Diseases

基　　金：山东省自然科学基金项目(ZR2014CM047);山东省自然科学青年基金项目(ZR2014CQ010);山东省科技攻关项目(2016GNC110032);江苏省兽用生物制药高技术研究重点实验室开放课题(JSKLKF1405);山东省自然科学基金(ZR201702230155)。

摘　　要：RT-PCR获得猪繁殖与呼吸综合征病毒(PRRSV)XZ06a株基因组,在基因组5’末端、3’末端分别添加锤头样核酶、丁型肝炎病毒核酶序列,并插入改造的pCAGEN表达载体中,构建DNA-launched感染性克隆,然后在病毒基因组ORF1b与ORF2间引入ORF6 TRS序列、酶切位点及EGFP基因,转染Marc-145细胞拯救表达EGFP的重组病毒并噬斑克隆纯化,Western blot检测EGFP表达并对获得的表达EGFP的重组病毒进行TCID50测定与分析。结果显示:PRRSV基因组大小为15145 nt,转染后5~7 d即可观察到重组病毒所致特异性病变灶,经过噬斑纯化,能够观察到携带EGFP基因的重组病毒在对应病变灶均有绿色荧光,最终获得稳定表达EGFP的重组病毒,且重组病毒感染16 h时即可检测EGFP表达;与亲本病毒相比,表达EGFP重组病毒致细胞出现病变时间有所延长,增殖滴度有所降低。本研究成功构建了表达EGFP的重组PRRSV,为开展PRRSV病毒复制机制、感染特性、致病机制、疫苗开发等研究奠定了基础。The whole genome of PRRSV XZ06a strain was obtained by RT-PCR and the hammerhead-like ribozyme(HHRz)and the Hepatitis delta virus ribozyme(HDVRz)sequences were added to its 5’and 3’ends.Then the modified genome was inserted into the modified pCAGEN expression vector to construct a DNA-launched infectious clone.The ORF6 TRS sequence and restriction enzyme cleavage sites were introduced between ORF1b and ORF2,and the EGFP gene was cloned and inserted,and finally the recombinant infectious clone was transfected into Marc-145 cells to rescue the virus expressing EGFP.The virus plaques were purified to determine their\proliferation titers and the EGFP expression at different time points.The results showed that the PRRSV genome length was 15,145 nt,and the rescued recombinant viruses developed CPE at days 5-7 post transfection.The recombinant virus carried the EGFP gene as the green fluorescence was observed in the corresponding CPE sites.The EGFP was detected at 16 h post infection.The recombinant virus had lower titer than the parental virus and developed CPE later.In this study,the recombinant PRRSV expressing EGFP was constructed,which laid a foundation for the further study of PRRSV infection and replication,pathogenic mechanism and vaccine development.

关 键 词：猪繁殖与呼吸综合征病毒 感染性克隆 ORF6转录调控序列 EGFP 噬斑纯化 

分 类 号：S852.659.6[农业科学—基础兽医学]