PRRSV感染性克隆5′非翻译区序列缺失突变分析  

5′ UTR deletion analysis of porcine reproductive and respiratory syndrome virus

作　　者：高飞[1] 姚火春[2] 王金勇[1] 余丹丹[1] 袁世山[1] 

机构地区：[1]中国农业科学院上海兽医研究所／中国动物卫生与流行病学中心上海分中心动物传染病研究室农业部动物寄生虫病重点实验室,上海200232 [2]南京农业大学动物医学院,江苏南京210095

出　　处：《中国兽医学报》2009年第1期25-29,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金重点项目(30530580);科技部重大基础研究“973”专项(2005CB523202)

摘　　要：在PRRSV感染性克隆pCBC2的基础上,通过突变PCR获得了病毒基因组5′UTR系列缺失的全长突变体克隆:pCBCM1、pCBCM3、pCBCM5、pCBCM7、pCBCM9、pCBCM45、pCBCM69、pCBCM100,随后将上述突变体体外转录成RNA之后转染MARC-145细胞,进行病毒感染性、复制和转录分析,以鉴定特定的缺失区的调控功能。结果显示,病毒5′UTR起始的第1个碱基是保持病毒感染性非必需的核苷酸;前45个碱基对病毒基因组的复制是非必需的。Northern-Blot结果显示pCBCM1亚基因组mRNA可以进行高效率的转录,而其余突变体的亚基因组转录受到严重抑制。由此证明,PRRSV 5′UTR的完整性对病毒意义重大,碱基的少量以及大部分缺失会对病毒产生极大的影响。The genomic 5＇ untranslated region （UTR） of single-stranded,positive-sense RNA viruses is thought to play a very important role in viral genomic replication,subgenomic mRNA transcription and protein translation. In this study,based on the porcine reproductive and respiratory syndrome virus （PRRSV） infectious full length eDNA elone pCBC2,a series of eDNA clones with 5＇ UTR truncated were obtained through PCR-based mutagenesis. Then the RNAs via in vitro transcription were respectively transfected into MARC-145 cells for further cytopathie effect （CPE） observation,genomic and subgenomic mRN A analysis,to investigate the regulatory function of 5＇ UTR. Results showed that the first base pair of PRRSV 5＇ UTR should be dispensable for the virus infection; the 45bp in 5＇ end of 5＇ UTR was dispensable for genomic RNA replication but those mutants truncated in this region couldn＇t cause CPE. The results mentioned above indicated that the integrity of 5＇ UTR should be crucial for the replication and transcription of PRRSV.

关 键 词：猪繁殖与呼吸综合征 感染性克隆 体外转录 缺失突变 反向遗传系统 

分 类 号：S852.65[农业科学—基础兽医学]