靶向狂犬病病毒N基因shRNA抑制狂犬病病毒复制的研究

为探讨小干扰RNA(siRNA)对狂犬病病毒(RV)复制的干扰作用,以RVN基因为靶向目标,设计合成4对编码siRNA的DNA序列,然后分别连接到载体pRNATU6.3-Hygro上,转染BHK细胞,在潮霉素-B抗性压力下筛选出4个阳性细胞株(BHK-N1、BHK-N2、BHK-N3和BHK-N4)。用100TCID50CVS-11毒分别感染24孔板内的上述细胞,利用Real-time RT-PCR、半数细胞培养物感染量(TCID50)、直接免疫荧光和Western blot检测抑制效率。接毒后48 h,在BHK-N1、BHK-N2、BHK-N3、BHK-N4细胞株中,Real-time RT-PCR检测到各细胞株均在不同程度上抑制了RV的增殖,其中BHK-N2、BHK-N1抑制效率高,分别为99%、67.72%,而BHK-N3、BHK-N4仅为38.59%和11.33%,抑制效果较弱;TCID50检测病毒数量比空载体表达细胞毒价分别低24、96.2、2.14和1.1倍;直接免疫荧光检测表明,4株细胞中RV含量为病毒对照的31%、1%、54%、82%,即BHK-N1、BHK-N2对病毒的沉默作用较强;Western blot结果显示BHK-N1、BHK-N2细胞株中RV N蛋白条带明显减弱。4种检测结果均表明BHK-N1和BHK-N2能有效干扰RV的复制。本研究在细胞水平筛选出具有高效抑制RV复制的2个靶位点N-489和N-701,为RV的基因功能研究、抗病毒药物的开发奠定了基础。

Detection of the Interference to the Replication of Rabies Virus by BHK Cell Strains Transcribing shRNA Targeted to the N Gene

In this study,the replication of rabies virus(RV) was inhibited by RNA interference in vitro experimentally in order to accumulate essential data of the application of RNAi in the researches of RV genomic function and the post-exposure treatment of rabies.Targeting N gene of RV,four shRNA expression plasmids were designed and constructed based on the vector pRNATU-6.3-Hygro which expresses fusion protein of green fluorescent protein as a report gene.Four cell strains(BHK-N1,BHK-N2,BHK-N3,BHK-N4) expressing ...