LE-PolyICLC在小鼠感染模型抗登革热Ⅱ型病毒的研究
发布时间：2019-05-17 16:48
【摘要】：登革热是由登革热病毒(Dengue viruses, DENVs)引起的一种急性热性传染病。近年来,随着登革热流行的日益严重,对登革热病毒进行有效的防治是全世界共同关注的问题。目前并无临床可用的特异性预防与治疗药物。近期登革热疫苗Dengvaxia(?)首获墨西哥当局批准,成为全球第一个登革热疫苗产品,但该疫苗还尚未在全球推广和应用。本研究成功建立了两个检测方法和一个小鼠感染模型。并以此为基础,首次在BABL/c小鼠模型开展核酸类药物LE-PolylCLC (Liposome-PolylCLC)抗登革热Ⅱ型病毒感染的研究。本研究共分为四个部分：一、登革热Ⅱ型病毒感染小鼠血清抗体的检测提取并扩增DENV2 NGC毒株EⅢ基因片段,将其克隆至原核表达载体2EASY(?)-Blunt E1。在大肠杆菌BL21(DE3)中成功表达出大小约为17 KD的EⅢ重组蛋白,将以包涵体形式表达的蛋白使用稀释复性的方法进行复性,浓缩,过柱纯化。将纯化的重组蛋白免疫小鼠,获得EⅢ重组蛋白的多克隆抗体血清。间接免疫荧光显示获得的多克隆抗体血清可与DENV2病毒发生特异性反应。进一步应用纯化的EⅢ重组蛋白建立了间接ELISA方法。并使用该方法对DENV2感染小鼠血清样本的抗体水平进行检测。二、登革热Ⅱ型病毒感染小鼠脑组织病毒滴度的测定根据DENV2 NGC毒株E基因序列,设计一对特异性引物和TaqMan探针。建立了DENV2 TaqMan实时荧光定量PCR检测方法。以构建的2EASY-T1-E重组质粒为标准品,得到标准曲线,其R值为0.98,Eff值为122.53%,说明该方法比较准确可靠。该方法的最小检测量为100拷贝／反应,具有较高的灵敏性。批内和批间样品的变异系数分别为0.53～1.90%和0.85～2.54%,该方法重复性也比较好。进一步使用该方法对DENV2感染小鼠脑组织样本的病毒含量进行测定。三、登革热Ⅱ型病毒小鼠感染模型的建立成功建立了DENV Ⅱ型病毒NGC毒株BABL/c小鼠脑内注射感染模型。NGC毒株以1×106pfu的病毒量脑内注射感染BABL/c小鼠。小鼠被病毒感染后体重下降并呈现出后肢瘫痪、皮肤出血等临床发病症状,感染后第9天症状最为严重并出现死亡,并且小鼠脑组织存在明显的病理损伤。对小鼠脑组织的病毒含量进行测定,发现病毒可在小鼠脑部复制和增殖,并在感染后第9天达到峰值。小鼠还能够产生相应的抗体来中和病毒,在感染后第21天抗体水平达到最高。四、LE-PolylCLC在小鼠模型中抗登革热Ⅱ型病毒感染的研究按1 mg/kg的用药剂量静脉注射LE-PolylCLC能够提高感染DENV2小鼠的存活率,减轻临床发病症状及对脑组织的病理损伤。LE-PolylCLC能够有效抑制DENV2在小鼠脑部的增殖,Western blotting以及间接免疫荧光数据也进一步验证了该结论。LE-PolylCLC通过静脉注射给药途径发挥的药效要优于其它给药途径。LE-PolylCLC药效发挥可能主要通过上调与天然免疫相关的IFN-y等细胞因子的表达,并且小鼠血清中抗体水平也有一定程度的升高。另外,LE-PolyICLC在该模型中抗DENV2的效果要优于PolylCLC。
[Abstract]:Dengue fever is an acute febrile infectious disease caused by Dengue virus (DENVs). In recent years, with the increasing prevalence of dengue fever, the effective prevention and treatment of dengue virus is a common concern of the whole world. Currently, there is no clinically available specific prevention and treatment drug. Dengue vaccine, Dengvaxia (? ) The first Mexican authorities have been approved by the Mexican authorities to be the world's first dengue vaccine product, but the vaccine has not yet been extended and applied globally. Two detection methods and a mouse infection model were successfully established in this study. On the basis of this, the first time in the BABL/ c mouse model, the study of the anti-dengue type 鈪，
本文编号：2479256
[Abstract]:Dengue fever is an acute febrile infectious disease caused by Dengue virus (DENVs). In recent years, with the increasing prevalence of dengue fever, the effective prevention and treatment of dengue virus is a common concern of the whole world. Currently, there is no clinically available specific prevention and treatment drug. Dengue vaccine, Dengvaxia (? ) The first Mexican authorities have been approved by the Mexican authorities to be the world's first dengue vaccine product, but the vaccine has not yet been extended and applied globally. Two detection methods and a mouse infection model were successfully established in this study. On the basis of this, the first time in the BABL/ c mouse model, the study of the anti-dengue type 鈪，
本文编号：2479256
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