畜牧兽医学报2018，Vol.49Issue(3)：637-643,7.DOI:10.11843/j.issn.0366-6964.2018.03.022
猪繁殖与呼吸综合征病毒类NADC30株CHsx1401感染性克隆的构建
Construction of an Infectious cDNA Clone of Porcine Reproductive and Respiratory Syndrome Virus NADC30-like CHsx1401
摘要
Abstract
Porcine reproductive and respiratory syndrome virus (PRRSV) remains the most economically important pathogen impacting swine industry worldwide.Recently,the novel NADC30-like strains of PRRSV resulting in the re-emerging of clinically PRRS outbreaks has been characterized in China.In order to provide a basis for research on the mechanisms in relation to the genetic variation and evolution of PRRSV NADC30-like strain,CHsx1401,as well as the differential pathogenesis between this virus and other PRRSV strains,a full-length cDNA clone of NADC30-like CHsx1401 was generated.Four fragments of the whole genome of CHsx1401 were amplified by RT-PCR,and each was cloned into pJET1.2 blunt,and each clone was then ligated into the modified low-copy-number vector pWSK29 to generate the full-length cDNA clone,plasmid pWSK-CHsx1401.To distinguish the rescued virus from its parental virus,a restriction enzyme site Asc Ⅰ was introduced between fragment C and D through synonymous mutation (T11582G) as a genetic marker.Subsequently,the full-length eDNA clone was transfected into MARC-145 cells using Lipofectamine LTX and plus reagents to rescue the virus.As the results showed,the full-length eDNA clone (plasmid pWSK-CHsx1401) was constructed successfully,cytopathic effect (CPE) typical of PRRSV in MARC-145 cells could be observed at the second passage.The virus could be rescued from the plasmid pWSK-CHsx1401 by IFA,RT-PCR and sequencing.The rescued virus could be passaged stably in MARC-145 cells,and showed a similar growth curve to its parental isolate in vitro,with slightly lower titers at some time points than CHsx1401 in MARC-145 cells.Our results indicate that the full-length cDNA clone of the PRRSV NADC30-like CHsx1401 is infectious and viable virus can be rescued.The rescued virus (RvCHsx1401) exhibited similar growth ability to its parental virus in vitro.Our present study provides a platform for further research on the molecular mechanisms in relation to the variation and evolution of PRRSV NADC30-like and its differential pathogenesis from other strains of PRRSV.关键词
猪繁殖与呼吸综合征病毒/类NADC30/全长cDNA克隆/感染性/拯救Key words
porcine reproductive and respiratory syndrome virus (PRRSV)/NADC30-like/full-length cDNA clone/infectious/rescue分类
农业科技引用本文复制引用
卞婷,周磊,宋江伟,盖新娜,郭鑫,韩军,杨汉春..猪繁殖与呼吸综合征病毒类NADC30株CHsx1401感染性克隆的构建[J].畜牧兽医学报,2018,49(3):637-643,7.基金项目
国家自然科学基金重大项目(31490603) （31490603）
国家重点基础研究发展计划(2014CB542700) （2014CB542700）
国家生猪产业技术体系(CARS-35) （CARS-35）
