猪繁殖与呼吸综合征重组病毒非必需基因区的鉴定

The non-essential gene locus identification of recombinant porcine reproductive and respiratory syndrome virus

为鉴定猪繁殖与呼吸综合征病毒(PRRSV)复制的非必需基因区,本研究在高致病性PRRSV细胞致弱株感染性克隆Hu N4-F112的基础上,在其基因组ORF1b和ORF2a之间插入增强型绿色荧光蛋白(EGFP)基因,并在外源基因3'端下游插入该病毒的转录调控序列6(TRS6),构建PRRSV-EGFP感染性克隆。将其体外转录制备的病毒全长RNA转染Marc-145细胞后,拯救获得了表达EGFP的重组病毒(r Hu N4-F112-EGFP)。将r Hu N4-F112-EGFP在Marc-145细胞中进行连续传代至20代,其外源gfp基因仍能够持续表达,表明该重组病毒具有良好的遗传稳定性。与亲本病毒Hu N4-F112相比,重组病毒在病毒滴度、空斑形态等方面差异不显著;病毒生长曲线的结果显示,重组病毒具有良好的增殖能力。本研究结果表明,在PRRSV的ORF1b和ORF2a基因区中插入外源基因并未影响该病毒体外复制,该非必需基因区的鉴定为进一步研究以PRRSV疫苗为活病毒载体多价重组疫苗的研发奠定基础。

To identify the non-essential gene locus of porcine reproductive and respiratory syndrome virus （PRRSV）, the report gene of enhanced green fluorescent protein （EGFP） gene was inserted between ORFlb and ORF2a of the infectious clone of attenuated PRRSV vaccine strain HuN4-F112 by SOE PCR （designated pHuN4-F112-EGFP）, and the recombinant PRRSV （rHuN4-F112-EGFP） expressing EGFP was rescued by transfecting the full genomic viral RNA into Marc-145 cells which was prepared by in vitro transcrption from pHuN4-F112-EGFP, Then, genetically stable test showed that the foreign gene of gfp was stably expressing in rHuN4-F112-EGFP even passing in Marc-145 cells for 20 passages. Moreover, the viral titer and plaque morphology of the recombinant virus was similar to the parental virus, and the growth curves were also similar between the recombinant and parental virus. These results demonstrated that the insertion of gfp gene in the gene locus between ORFlb and ORF2a of PRRSV had no impacted on the virus replication in vitro, and the identification of the non-essential gene locus provided the basis for further development of the recombinant vaccine virus based on the PRRSV attenuated vaccine strain HuN4-F112.