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HSV-1是一种嗜神经病毒,能引起一系列神经系统严重症状,然而目前抗HSV-1药物易反弹、不能完全清除潜伏的病毒。ICP4对HSV复制、转录起主要调节作用,决定着溶细胞型感染或潜伏状态的平衡点。为了探寻新的抗病毒策略,本课题以HSV-1ICP4基因为靶点,设计合成2对siRNA,并构建重组真核慢病毒表达质粒pL-KO-puror-hU6-siRNA,通过脂质体转染和嘌呤霉素筛选建立靶向ICP4的4个siRNA单克隆细胞系,Real-timePCR法检测细胞系中ICP4的mRNA表达水平,TCID50法检测siRNA对HSV-1病毒复制能力的影响。结果显示靶向siRNA能有效抑制单克隆细胞系中的ICP4表达,并且抑制ICP4的表达后HSV-1病毒复制能力明显减弱,表明靶向ICP4的siRNA对HSV-1复制有明显抑制作用,且多位点siRNA联合干扰对病毒复制有协同抑制效果,有望应用于生物抗病毒药物的制备。
HSV-1 is a neurotropic virus that causes serious neurological symptoms. However, HSV-1 is currently susceptible to rebound and does not completely eliminate the latent virus. ICP4 on HSV replication, transcription plays a major regulatory role, determines the cytosol infection or latent state balance point. In order to explore a new antiviral strategy, we designed and synthesized two pairs of siRNA targeting HSV-1ICP4 gene and constructed the recombinant eukaryotic lentiviral expression plasmid pL-KO-puror-hU6-siRNA. And puromycin. Four siRNA monoclone cell lines targeting ICP4 were established. Real-time PCR was used to detect the mRNA expression of ICP4 in the cell lines. The TCID50 method was used to detect the effect of siRNA on HSV-1 virus replication. The results showed that targeting siRNA could effectively inhibit the expression of ICP4 in the monoclonal cell line and inhibit the replication of HSV-1 virus significantly after inhibiting the expression of ICP4, which indicated that siRNA targeting ICP4 had a significant inhibitory effect on HSV-1 replication, and more Site siRNA co-interference with virus replication synergistic inhibitory effect, is expected to be applied to the preparation of biological antiviral drugs.