摘要
反向遗传系统已成为研究猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndromevirus,PRRSV)结构与功能非常重要的手段,并且在设计基因工程疫苗中发挥不可替代的作用。因而,有效提高感染性克隆拯救病毒的效率和稳定性以及降低经济成本是一个急需解决的课题。本研究在已构建完成的高致病性PRRSV细胞传代毒株反向遗传系统(pAJXM)的基础上,做了以下三方面的工作:(1)将T7启动子换成hCMV(人类巨细胞病毒,Human cytomegalovirus)启动子,从而全长cDNA克隆不需经过体外转录成mRNA的过程,直接通过DNA转染MARC-145细胞拯救病毒;(2)研究hCMV启动子的TATA框与病毒基因组5末端之间最佳的碱基数目,使体内转染获得的病毒基因组5末端序列为病毒的真实序列;(3)在病毒基因组3末端添加丁型肝炎病毒核酶(delta hepatitis virus ribozyme,HDVr)序列,体内转染获得的病毒基因组3末端的非病毒序列通过核酶自动去除。研究发现,基于DNA转染的反向操作系统是可行的,并且hCMV启动子的TATA框与病毒基因组5末端之间的碱基数目设置为25和在病毒基因组3末端添加HDVr序列后,能够有效地提高全长感染性cDNA克隆的病毒拯救效率和稳定性。
Reverse genetics system has become an important method for dissecting the relationship between structure and function of porcine reproductive and respiratory syndrome virus（PRRSV） genes and played a significant role in development of genetic engineering vaccine.Therefore,the virus rescue efficiency and stability of reverse genetic system are badly in need of improvement.In this study,some work was conducted to improve rescue efficiency of reverse genetics system,including followings：（1） the T7 promoter with human cytomegalovirus（hCMV） promoter was replaced so that PRRSV could be rescued by directly DNA transfection;（2） the optima distance between TATA box of hCMV promoter and 5 terminal of viral genome was dissected in order to get the authentic 5 terminal sequence of viral genome;（3） a delta hepatitis virus self-cleaving ribozyme element was inserted following 3 terminal of viral genome in order to get the authentic 3 terminal sequence of viral genome.Results showed that the improved DNA-launched PRRSV reverse genetics system improved virus rescue efficiency.
出处
《中国动物传染病学报》
CAS
2010年第3期13-20,共8页
Chinese Journal of Animal Infectious Diseases
基金
"973"重大专项(2005CB523200)
上海市浦江人才计划(06PJ14118)
关键词
猪繁殖与呼吸综合征病毒
反向遗传系统
人类巨细胞病毒启动子
丁型肝炎病毒核酶
Porcine reproductive and respiratory syndrome virus（PRRSV）
reverse genetics system
humancytomegalovirus（hCMV）promoter
delta hepatitis virus ribozyme（HDVr）
