摘要
【目的】通过靶向PRRSV基因组中的M基因的siRNA来抑制PRRSV在Marc145细胞中的复制。【方法】构建4个能转录小发夹RNA(shRNA)的质粒,将其与靶蛋白表达质粒共转染HEK293A细胞,观察荧光或进行半定量PCR;或将其转染Marc145细胞,感染PRRSV后进行IFA、TCID50和实时PCR检测。【结果】shRNA表达质粒对M融合蛋白表达的抑制率约为50%,使M真核质粒表达蛋白的mRNA水平降低54%～64%,表达的shRNA在PRRSV感染后48h使病毒的TCID50和mRNA水平均降低到1/10～1/100倍,间接免疫荧光结果表明shRNA表达质粒转染孔的荧光细胞数显著减少。【结论】shRNA表达质粒特异性的抑制了靶蛋白M和PRRSV的复制,靶向PRRSV基因组M基因不同区域的siRNA可以作为控制该病毒传播的候选策略。
[Objective] To evaluate the inhibition ability of siRNAs targeting to M protein gene of porcine reproductive and respiratory syndrome virus. [Method] shRNA-expressing plasmids were constructed and delivered to HEK293A or Marc145 cells, efficiency of RNA interfering was assayed by semiquantitative RT-PCR, indirect immunofluorescence assay （IFA）, virus titre of TCID50 and real time PCR. [Result] After cotransfection with fusion-protein expressing plasmids, the fluorescence in HEK293A cells treated by shRNA expressing plasmids became obviously weaker compared to those cotransfected with target gene expressing plasmid and pSUPER plasmid. Different shRNA expressing vectors were also cotransfected into HEK293A cells with vectors expressing M proteins of PRRSV, and the mRNA level of M protein was inhibited by 54%-64% assayed by semi-quantitative PCR. These plasmids expressing shRNA were also delivered into Marc145 cells. After infection, these cells showed a significant decrease in virus yield when compared to control cells, by detection using virus titers （TCID50）, IFA and real-time RT-PCR. [Conclusion] siRNA targeting to M protein gene of PRRSV could inhibit M protein expression and PRRSV replication in Marc145 cells.
出处
《中国农业科学》
CAS
CSCD
北大核心
2008年第1期259-264,共6页
Scientia Agricultura Sinica
基金
国家自然科学基金(30471288、30270990)
江苏省自然科学基金(BK2005093)
教育部博士点基金项目(20030307012)
