逆转录病毒载体RNAi技术抑制猪瘟病毒在猪胚胎成纤维细胞的增殖

Inhibition of Classical Swine Fever Virus Replication by Retrovirus-delivered shRNA in Porcine Fetal Fibroblasts

利用针对猪瘟病毒(Classical swine fever virus,CSFV)石门株Npro基因mRNA的shRNA逆转录病毒表达载体,转导猪胚胎成纤维细胞,在G418的筛选压力下,获得7个稳定整合shRNA表达构件的猪胚胎成纤维细胞株。以100TCID50的CSFV分别感染96孔板内的上述细胞克隆,72h后以对感染细胞克隆进行间接免疫荧光分析及子代病毒滴度检测,结果显示,在所获得的7个抗性细胞株中,有3株细胞上猪瘟病毒的增殖显著降低,表明所构建的针对猪瘟病毒Npro基因mRNA的shRNA逆转录病毒整合细胞基因组后转录产生的siRNA可以有效抑制CSFV的复制。

In this study, the recombinant retrovirus which contains the CSFV-specific shRNAs （small hairpin RNA） targeting N^pro transcripts of classical swinefever virus was transfected into porcine fetal fibroblasts to drive expression of the CSFV-specific short hairpin RNA （shRNA）, After stable selection, G418-resistant porcine fetal fibroblasts clones were established. Integration of the vector was proven by PCR; the viral proliferation within the cells was examined by indirect immunofluorescence assay and determination of virus titration respectively. The levels of replication of CSFV were dramatically decreased by this RNAi system in porcine fetal fibroblasts stablely transfected with the vector. Our results may be useful for therapy in CSFV and other viral infections and provide new clues for animal breeding or establishing feasible antiviral animal models.