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  

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

作　　者：李红梅[1,2] 贺聪[2] 张茂[3] 孟轩[2] 张建峰[2] 张健騑[2] 郭鹏举[2] 

机构地区：[1]华南农业大学动物科学学院 [2]广东省农业科学院动物卫生研究所公共实验室 [3]广东省农业科学院动物科学研究所

出　　处：《中国畜牧兽医》2013年第12期12-20,共9页China Animal Husbandry & Veterinary Medicine

基　　金：农业部948项目(2011-Z56)

摘　　要：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-U6neo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。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.

关 键 词：猪繁殖与呼吸综合征病毒 RNA干扰 抗猪繁殖与呼吸综合征病毒细胞株 

分 类 号：S852.43[农业科学—基础兽医学]