表达猪圆环病毒2型ORF2基因的重组猪繁殖与呼吸综合征病毒的构建与鉴定  

Construction and Identification of a Recombinant PRRSV Expressing ORF2 of Porcine Circovirus Type 2

作　　者：张挺杰[1] 刘星[1] 孙涛[1] 朱雪蛟[1] 范宝超[1] 白娟[1] 姜平[1] 

机构地区：[1]南京农业大学农业部动物细菌学重点实验室,南京210095

出　　处：《病毒学报》2015年第1期65-73,共9页Chinese Journal of Virology

基　　金：教育部科技项目(313031;20120097110043);农业部行业专项子项目(201203039);国家生猪产业技术体系专项(CARS-36);国家自然基金(31230071)

摘　　要：猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)和猪圆环病毒2型(Porcine circovirus type 2,PCV2)是目前危害世界养猪业的两种重要病原。本研究采用PCR方法构建PRRSV疫苗株TJM-F92全长cDNA克隆载体pCMV-TJM,并在其ORF7和3′UTR之间插入AflⅡ/MluⅠ酶切位点和转录调控序列TRS6,构建获得pCMV-TJM-TRS表达载体。将PCV2ORF2基因插入该载体AflⅡ/MluⅠ位点,获得重组质粒pCMV-TJM-Cap。将pCMV-TJM、pCMV-TJM-TRS和pCMV-TJM-Cap分别转染Marc-145细胞,拯救获得3种重组病毒rTJM、rTJM/TRS和rTJM/Cap。基因测序列、酶切鉴定、Western blot、间接免疫荧光和病毒生长特性结果显示,3种重组PRRSV病毒都含有特征性分子标记,在Marc-145细胞增殖特性与亲本病毒相似;rTJM/Cap传至第8代,仍含有外源Cap基因,病毒感染细胞能有效表达PCV2Cap蛋白,从而为PRRSV致病机制和PRRSV-PCV2疫苗研究奠定了重要基础。Porcine reproductive and respiratory syndrome virus（PRRSV）and porcine circovirus type 2（PCV2）are very two important pathogens that have coursed huge economic losses in swine production in worldwide.In this study,a vector pCMV-TJM containing the full-length cDNA clone of PRRSV attenuated strain TJM-F92 was firstly constructed by PCR method.Then a gene sequence containing AflⅡ/MluⅠ e restriction enzyme sites and a transcription regulatory sequence for ORF6（TRS6）was inserted between ORF7 and 3′UTR,yielding a expression vector pCMV-TJM-TRS.Subsequently,aplasmid pCMVTJM-Cap was constructed by cloning of PCV2ORF2 gene into the unique sites AflⅡ/MluⅠ of pCMVTJM-TRS plasmid DNA.Then three recombinant PRRSV,rTJM,rTJM/TRS and rTJM/Cap,were rescued by transfection of pCMV-TJM,pCMV-TJM-TRS and pCMV-TJM-Cap into Marc-145 cells,respectively,and confirmed by the genome sequence,restriction enzyme digestion,Western Blot and IFA.They all had the molecular markers which was different from the parent virus.The growth characteristics of the rescued viruses were similar to that of parent virus.rTJM/Cap could also express efficiently PCV2 Cap protein in Marc-145 cells.At passage 8,it still had PCV2ORF2 gene which examined by RT-PCR.It indicated that the full-length cDNA clone of PRRSV attenuated strain TJM-F92 and recombinant PRRSV rTJM/Cap expressing PCV2 Cap protein were successfully constructed.It made an important foundation for studying on the pathogenic mechanisms of PRRSV and PRRSV-PCV2 vaccine in the future.

关 键 词：猪繁殖与呼吸综合征病毒 猪圆环病毒2型 反向遗传 重组病毒 

分 类 号：S852.65[农业科学—基础兽医学]