shRNA抑制猪繁殖与呼吸综合征病毒(PRRSV)JXA1-R株在Marc-145细胞中复制及抗PRRSV细胞株的建立

猪繁殖与呼吸综合征（porcine reproductive and respiratory syndrome，PRRS）是世界上主要的猪传染性疾病之一，目前，疫苗和抗病毒药物只能提供有限的保护。本研究选择猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus，PRRSV）JXA1-R株ORF1a、ORF3、ORF4、ORF5、ORF6、ORF7的保守区域为靶序列，利用pSilencer2.1-U6 neo构建shRNA表达载体并转染Marc-145细胞。经实时荧光定量PCR、细胞病变及病毒滴度分析，结果显示，ORF1a-4-shRNA、ORF3-1-shRNA、ORF6-2-shRNA干扰质粒能明显抑制病毒基因的转录，并有效抑制PRRSV在Marc-145上的增殖，显著降低病毒滴度。最后筛选ORF6-2-shRNA表达质粒转染猪胎儿成纤维细胞，构建稳定转染细胞株，为抗PRRSV转基因猪的生产奠定了基础。

Inhibition of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) JXA1-R Replication in Marc-145 Cells by shRNA and Establishment of Anti-PRRSV Cell Lines

Porcine reproductive and respiratory syndrome (PRRS) was one of the most important infectious diseases of swine in the world.However,current vaccination strategies and antiviral drugs provided only limited protection. In this study, the ability of specific short hairpin RNA targeting to ORF1a, ORF3, ORF4, ORF5, ORF6, ORF7 were constructed, and it was found that ORF1a-4-shRNA, ORF3-1-shRNA, ORF6-2-shRNA could effectively down-regulate specific gene expression and inhibit viral replication in Marc-145 cells compared to the controls. In addition, in this study ORF6-2-shRNA was selected to be transfected into porcine fetal fibroblasts to establish anti-PRRSV cell lines. This work might provide the foundation for further research on the production of anti-PRRSV transgenetic pig.