猪繁殖与呼吸综合征病毒强、弱毒株Nsp9基因的反向遗传学替换改造

摘要

猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)Nsp9基因编码RNA依赖的RNA聚合酶,在病毒复制中发挥重要作用.为了探索Nsp9基因与病毒的毒力、复制能力及细胞嗜性的相关性,本研究利用反向遗传学技术将PRRSV弱毒疫苗毒CH-1R的Nsp9基因进行克隆,酶切后连接到强毒株XH-GD的感染性克隆上,进行病毒的拯救.结果表明,成功构建替换了CH-1R Nsp9基因的重组病毒.对重组病毒的生物学特性研究发现,拯救病毒的生长速度和病毒滴度明显高于亲本毒株rXH-GD,但是低于疫苗毒CH-1R,重组毒株的噬斑大小与CH-1R相似,初步推测疫苗株CH-1R的聚合酶有助于提高病毒的滴度.本试验为进一步研究Nsp9基因对病毒毒力的影响奠定基础.%The Nsp9 gene of porcine reproductive and respiratory syndrome virus (PRRSV) coded the RNA polymerase,which played an important role in the virus replication.In order to determine the interaction between the Nsp9 gene and virulence,replication capacity and cell tropism,the Nsp9 gene sequence of PPRSV vaccine strain CH-1R was cloned,and ligated to a virulent strain XH-GD infectious clone after restriction enzyme digestion,and transformed the virus under the basis of the reverse genetics.The results showed that the virus was successfully saved.And the results of biological characteristics test showed that titers and growth rate of saved virus were obviously higher than that of parental strain rXH-GD,but were lower than vaccine strain CH-1R.While plaques of two strains were similar in size.The polymerase of vaccine strain CH-1R could help to improve the drop degree of the virus,and this test could lay the foundation for further study of the effect of Nsp9 gene on virus virulence.