小鼠体内SEG蛋白靶向运送shRNA抑制狂犬病毒复制

Targeted inhibition of Rabies virus gene expression by a chimeric multidomain protein mediated shRNA delivery

摘要:

【目的】本试验前期已经证实,用单链抗体(scFv)-绿脓杆菌跨膜区(ETA)-酵母DNA结合结构域(GAL4)表达的蛋白(简写为SEG蛋白), SEG能与含shRNA(short hairpin RNA)的质粒(pRNATU6.3-shRNA)结合形成复合物SEG-shRNA,并靶向运送该质粒进入感染狂犬病毒(Rabies virus, RV)的细胞,抑制RV复制。本研究用感染狂犬病病毒的小鼠模型,进行SEG-shRNA复合物小鼠体内靶向性运送siRNA(short interfering RNA)和抑制RV复制的研究。【方法】用已建立RV CVS-24株小鼠肌肉注射模型进行试验。取50 LD₅₀ CVS-24攻毒,在攻毒后12 h尾静脉注射SEG-shRNA,流式细胞仪检测SEG-shRNA的体内靶向性;同样方法攻毒后,尾静脉注射SEG-shRNA,连续4 d,攻毒后第5天小鼠脑组织用qRT-PCR、RT-PCR、Western blot、免疫荧光染色法检测其中RV的含量;统计小鼠存活率;并检测小鼠体内IFN-α含量,从而分析SEG-shRNA在体内的抗病毒作用。【结果】结果表明仅在RV攻毒小鼠的注射部位检测到绿色荧光蛋白(green fluorescent protein, GFP)的表达,未注射RV的腿部及脑、肝、脾、肾均无GFP表达,说明SEG-shRNA可靶向RV感染细胞运送shRNA。攻毒后第5天脑组织qRT-PCR结果表明靶向药物组比病毒对照减少4.88倍(3.9/0.8); RT-PCR、Western blot、免疫荧光染色试验结果表明使用SEG-shRNA组病毒量明显少于病毒对照组;且攻毒后13 d,动物存活率达50%,而病毒对照100%死亡。检测小鼠体内IFN-α未见升高。【结论】以上试验表明SEG蛋白在小鼠体内靶向运送含shRNA的质粒到感染组织细胞;对小鼠体内RV有明显抑制作用,因此可以用于狂犬病毒感染的特异辅助性救治研究。

Abstract:

[Objective] In this study, a new chimeric protein SEG expressed in previous work was applied to evaluate its translocating efficiency of shRNA to rabies virus infected cells in mice, meanwhile, the capability of anti-rabies virus was investigated.[Methods] Rabies virus strain CVS-24 was inoculated into the hind leg to establish a mouse model of rabies in a dose of 50 LD₅₀; 12 h thereafter the mice were injected intravenously with shRNA-producing plasmid mixed with SEG. To test shRNA delivery, single-cell suspensions from brain, spleen and liver were examined by flow cytometry. Rabies virus in brain tissue of mice was detected by qRT-PCR, RT-PCR, western blot and directed immunofluorescence assay. Mice were monitored for survival and serum samples were tested for IFN-α levels.[Results] No green fluorescent protein(GFP) was seen in the spleen or liver, suggesting that SEG allows specific targeting of RV-infected cells. RT-PCR and western blot showed that mice treated with SEG-shRNA had lower rabies virus RNA and protein levels than the controls. Real-time PCR showed that rabies virus was reduced 4.88 fold compared to the mock cells. Survival of RV-infected mouse showed a significant protection from rabies virus infection by SEG-shRNA treatment. The survival was up to 50% whereas the control group all died. IFN was not induced in SEG-shRNA treated animals.[Conclusion] shRNA-producing plasmid was specifically delivered into rabies virus infected cells using the SEG protein, and effectively inhibited rabies virus gene expression and replication in vivo. SEG-shRNA can be used for adjuvant treatment for rabies.