pSIREN/PB1 siRNA重组质粒抗甲型流感病毒研究

目的研究重组质粒pSIREN/PB1在鸡胚尿囊液中干扰甲型流感病毒复制的效果。方法设计1对编码PB1siRNA的寡核苷酸序列,经退火互补形成双链,再克隆至带有U6启动子的RNAi ready pSIREN-shuttle线性化载体,转化大肠杆菌JM109感受态细胞,双酶切和测序鉴定重组质粒pSIREN/PB1。大量提取重组质粒pSIREN/PB1,用脂质体进行包裹后,按每只鸡胚90μg质粒量接种于10日龄鸡胚尿囊腔,4血凝单位甲型流感病毒液分别于质粒接种后0h、24h、48h接种于鸡胚尿囊腔,流感病毒接种48h后收集无色澄清尿囊液进行血凝实验检测流感病毒效价。结果甲型流感病毒PB1基因的siRNA编码序列成功克隆至pSIREN质粒载体。经酶切和测序分析,重组质粒pSIREN/PB1中的siRNA编码序列均完全正确。将脂质体包裹的pSIREN/PB1质粒和病毒液同时注射入鸡胚尿囊腔后,收获的甲型流感病毒效价最低。结论成功构建了表达甲型流感病毒PB1siRNA的重组质粒pSIREN/PB1,并且它可以抑制鸡胚中流感病毒复制。此研究进一步证实了RNA干扰对流感病毒复制的抑制作用,为开发流感基因防治新方法奠定了基础。

Study on the recombinant plasmid pSIREN/PB1 to inhibit replication of influenza A virus in embrionated chicken eggs

Objective To research the effect of pSIREN/PB1 on inhibiting the influenza A virus in allantoic cavity of embrionated chicken eggs. Methods One pair of siRNA oligos were designed and synthesized, The oligos were anealed to form double DNA strands, The obtained products were cloned into the RNAi ready pSIREN-shuttle vector, containing U6 promoter, to form annular recombinant plasmid pSIREN/PB1, The pSIREN/PB1 was transformed into the JM109 E. coli, and identified by endonuclease digestion and sequence analysis. The pSIREN/PB1 was enveloped with liposome and then 90μg of the mixture was inoculated into allantoic cavity of each embrionated chicken egg. 0 h, 24 h, 48 h later, the 4 U influenza virus were inoculated respectively into the 10-day-old embrionated chicken eggs. Allantoic fluid was harvested 48 h after virus inoculation and the hemagglutination assays was carried to see the virus titer. Results The PBlsiRNA oligo was correctly cloned into the pSIREN/PB1. We found that inoculating influenza virus and recombinant plasmid with lipidosome at the same time could inhibit influenza A virus most effectively in the embrionated chicken eggs. Conclusion The recombinant plasmid pSIREN/PB1 could distinctly inhibit influenza A virus replication in the chicken eggs, RNA interference could inhibit the virus replication, and it offers a solid foundation for developing new methods for gene therapy and prevention against influenza.