小鼠体内SEG蛋白靶向运送shRNA抑制狂犬病毒复制

发布时间：2018-03-22 20:02

本文选题：狂犬病毒　切入点：shRNA　出处：《微生物学报》2016年01期 　论文类型：期刊论文

【摘要】：【目的】本试验前期已经证实,用单链抗体(sc Fv)-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sh RNA(short hairpin RNA)的质粒(p RNATU6.3-sh RNA)结合形成复合物SEG-sh RNA,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-sh RNA复合物小鼠体内靶向性运送si RNA(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 LD_(50) CVS-24攻毒,在攻毒后12 h尾静脉注射SEG-sh RNA,流式细胞仪检测SEG-sh RNA的体内靶向性;同样方法攻毒后,尾静脉注射SEG-sh RNA,连续4 d,攻毒后第5天小鼠脑组织用q RT-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-sh RNA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-sh RNA可靶向RV感染细胞运送sh RNA。攻毒后第5天脑组织q RT-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-sh RNA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sh RNA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] it has been confirmed that, A protein expressed in the transmembrane region of Pseudomonas aeruginosa (DNA binding domain GAL4) (SEG protein can bind to plasmid RNA(short RNATU6.3-sh RNAs containing sh RNA(short hairpin) to form complex SEG-sh RNAs, which is targeted to transport the plasmid into the sense of entry. The cells infected with rabies virus RV, Inhibition of RV replication. A mouse model infected with rabies virus was used in this study. The aim transport of si RNA(short interfering (si RNA(short interfering) and inhibition of RV replication in mice with SEG-sh RNA complex were studied. [methods] an intramuscular injection model of RV CVS-24 strain was established. SEG-sh RNAs were injected into tail vein 12 hours after poisoning, and the in vivo targeting of SEG-sh RNA was detected by flow cytometry. SEG-sh RNA was injected into caudal vein for 4 days. On the 5th day, the brain tissue of mice was detected by Q RT-PCRX RT-PCRX Western blot, the RV content was detected by immunofluorescence staining, the survival rate of mice was counted, and the content of IFN- 伪 in mice was measured. The antiviral effect of SEG-sh RNA in vivo was analyzed. [results] the results showed that the expression of green fluorescent protein (GFP) was detected only in the injection site of RV infected mice, and no expression of GFP was found in the legs, brain, liver, spleen and kidney of mice without RV injection. The results of Q RT-PCR in brain tissue showed that the target drug group was 4.88 times lower than that of the control group. The results of immunofluorescence staining showed that the amount of virus in the group treated with SEG-sh RNA was significantly lower than that in the control group. The survival rate of the mice was 50% and the virus control was 100%. IFN- 伪 was not increased in the mice. [conclusion] the results showed that the SEG protein could transport the plasmid containing sh RNA to the infected tissue cells in vivo. It can be used in the study of specific adjuvant treatment of rabies virus infection.
【作者单位】：青岛农业大学动物科技学院;军事医学科学院军事兽医研究所;吉林省人畜共患病预防与控制重点实验室;
【基金】：公益性行业(农业)科研专项(201103032,201303042)~~
【分类号】：S852.65

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