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目的设计针对H5N1高致病性禽流感病毒(HPAIV)核蛋白(NP)基因的干扰RNA(siRNA),研究其在细胞水平抑制病毒复制的效果。方法根据siRNA设计原则,设计并合成3对靶向HPAIV NP基因的siRNA,构建表达性质粒(ps-NP732、ps-NP863、ps-NP1344),分别转染MDCK细胞并攻毒,通过病毒滴度测定r、eal-time PCR、间接免疫荧光试验和Western blot,检测siRNA抑制AIV复制的效果。结果设计的3对siRNA中,ps-NP863明显抑制AIV复制,病毒滴度与对照组相比降低31倍,real-time PCR、间接免疫荧光试验、Western blot结果与病毒滴度测定结果相符,而ps-NP732和ps-NP1344无抑制AIV复制作用。结论构建的ps-NP863可显著抑制HPAIV复制,为开发抗AIV治疗制剂和动物机体抗AIV研究奠定了基础。
Objective To design an interfering RNA (siRNA) against the nucleocapsid protein (NP) gene of H5N1 HPAI virus and study its inhibitory effect on virus replication at the cellular level. Methods According to the principle of siRNA design, three pairs of siRNA targeting HPAIV NP gene were designed and synthesized. Expressing plasmids (ps-NP732, ps-NP863, ps-NP1344) were constructed and transfected into MDCK cells respectively. The r, eal-time PCR, indirect immunofluorescence assay and Western blot were used to detect the effect of siRNA on AIV replication. Results Among the three pairs of siRNAs designed, ps-NP863 significantly inhibited AIV replication and the virus titer was reduced by 31-fold compared with that of the control group. The results of real-time PCR, indirect immunofluorescence assay and Western blot were consistent with the results of virus titer assay Ps-NP732 and ps-NP1344 did not inhibit AIV replication. Conclusion The constructed ps-NP863 can significantly inhibit the replication of HPAIV, which lays the foundation for the development of anti-AIV treatment of anti-AIV and the study of animal anti-AIV.