猪繁殖与呼吸综合征病毒高致病性毒株HN07-1反向遗传操作平台的构建  

Construction of Reverse Genetic Platform of Highly Pathogenic Porcine reproductive and respiratory syndrome virus Strain HN07-1

作　　者：陈鑫鑫[1] 王林建[1] 乔松林[1] 李睿[1] 邓瑞广[1] 张改平[1,2] CHEN Xin-Xin;WANG Lin-Jian;QIAO Song-Lin;LI Rui;DENG Rui-Guang;ZHANG Gai-Ping(Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;Henan Agricultural University,Zhengzhou 450002,China)

机构地区：[1]河南省农业科学院动物免疫学重点实验室,郑州450002 [2]河南农业大学,郑州450002

出　　处：《农业生物技术学报》2021年第10期2043-2050,共8页Journal of Agricultural Biotechnology

基　　金：河南省农业科学院杰出青年科技基金(2020JQ06);河南省生猪产业体系创新团队项目(S2012-06);财政部和农业农村部:国家现代农业产业技术体系。

摘　　要：猪繁殖与呼吸综合征是世界规模化猪场的主要疫病之一,主要由猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)引起。本研究旨在构建PRRSV高致病性毒株HN07-1反向操作系统。通过将基因组进行分段克隆和测序的方法获得HN07-1毒株全长基因组序列。运用反向遗传学技术将全基因组分段克隆至载体上,构建含有全长HN07-1毒株cDNA的感染性克隆pcDNA3.2-rHN07-1,并以此为骨架,构建在PRRSV基因组ORF1b和ORF2a间插入增强绿色荧光蛋白(enhanced green fluorescent protein, EGFP)的重组克隆pcDNA3.2-rHN07-1-EGFP;将构建的质粒转染Marc-145细胞拯救病毒。采用免疫荧光实验(immune fluorescence assay, IFA)和Western blot进行表达鉴定并对重组病毒进行滴度测定与分析。结果表明,PRRSV毒株HN07-1基因组全长15 345 nt,转染Marc-145细胞盲传一代后培养48 h出现典型的细胞病变效应,IFA和Western blot检测结果表明,拯救病毒与亲本毒株HN07-1一致,都能检测到PRRSV N蛋白表达。并且拯救病毒与亲本毒株具有相似的生长动力特性。综上所述,本研究成功构建了HN07-1感染性克隆,可用于反向遗传操作,为PRRSV致病机制研究及疫苗研发提供了参考。Porcine reproductive and respiratory syndrome(PRRS), one of the leading outbreaks on a world scale in swine farms, is caused by Porcine reproductive and respiratory syndrome virus(PRRSV). This study aimed to construct reverse genetic system of highly pathogenic PRRSV(HP-PRRSV) strain HN07-1. The whole genome of HN07-1 was first obtained by a method in which the genome was segmently cloned and sequenced. Based on the reverse genetics technique, fragments of the whole genome were cloned into pcDNA3.1(+) vector and full-length infectious clone of HN07-1 strain, designated as pcDNA3.2-rHN07-1,were constructed. Besides, the recombinant plasmid pcDNA3.2-rHN07-1-EGFP was constructed by inserting EGFP between PRRSV ORF1 b and ORF2 a. The recombinant viruses were rescued by transfecting the constructed plasmids into Marc-145 cells and identified by immunofluorescence assay(IFA) and Western blot.The results showed that the full-length of HN07-1 genome was 15 345 nt. The typical cytopathic effect(CPE)was observed at 48 h of culture after one blind passage on Marc-145 cells. The results of IFA and Western blot indicated that PRRSV N protein expression of the rescued viruses and parent strain was confirmed. Moreover,the rescued viruses exhibit similar growth dynamic characteristics with its parent strain. Taken together, these results indicate that the infectious clones can be used for reverse genetic operation, which lays a foundation for the research of pathogenesis and vaccine development of PRRSV.

关 键 词：猪繁殖与呼吸综合征病毒(PRRSV) HN07-1 感染性克隆 反向遗传技术 病毒拯救 

分 类 号：S852.65[农业科学—基础兽医学]