亚马逊利什曼原虫无鞭毛体的体外培养及鉴定

发布时间：2019-05-13 18:27

【摘要】：目的:建立亚马逊利什曼原虫体外培养体系以获得大量的无鞭毛体供免疫学及分子生物学研究;进一步证明前鞭毛体转化的无鞭毛体的抗原特异牲、基因表达水平及对宿主的感染特性。方法:我们通过改良培养基,将小牛血清从20%提高到50%,在体外将前鞭毛体转化为无鞭毛体,并在体外纯培养。然后对三种不同来源的无鞭毛体(动物皮损来源、J774巨噬细胞感染来源及前鞭毛体转化)进行鉴定:用特异单克隆抗体的间接免疫荧光染色分析对不同来源无鞭毛体(P-4和P-8)及前鞭毛体(GP-46/M2)进行染色检测原虫特异性抗原的表达;设计了编码P-4、GP-46及β-微管蛋白的特异引物,用于RT-PCR分析;同时比较了三种来源的无鞭毛体的巨噬细胞感染特性。结果:三种来源的无鞭毛体经光镜检查显示相近的形态学特征;用特异单克隆抗体的间接免疫荧光染色分析法对三种不同来源的无鞭毛体(P-4和P-8)及前鞭毛体(GP-46/M2)进行鉴定。P-4和P-8单克隆抗体对三种无鞭毛体的染色呈阳性,而且浓度及部位相似。而前鞭毛体仅与P-8单克隆抗体(mAb)有较弱的阳性反应。P-4mAb则无阳性反应。相反,GP-46/M2单克隆抗体与前鞭毛体呈强阳性反应,却不与无鞭毛体出现强阳性反应;用RT-PCR分析三种来源的无鞭毛体均观察到P-4的特异性带,且浓度相似,而在前鞭毛体中则几乎测不到。相反,在前鞭毛体中则GP-46特异性带的高表达(325bp),三种来源的无鞭毛体却是弱表达。另外,三种来源无鞭毛体对巨噬细胞的感染率均接近100%。结论:通过温度改变(提高)将前鞭毛体转化为无鞭毛体后,将培养基小牛血清的浓度提高到50%,使无鞭毛体在体外纯培养并长期传代。建立了亚马逊利什曼原虫体外培养体系。三种来源的无鞭毛体在形态学上、在抗原特性方面、P-4基因水平上、对巨噬细胞感染物性方面都具有相同的特性。因此,从前鞭毛体转化而来的鞭毛体可作为良好的无鞭毛体的丰富来源。供进一步在免疫学等方面对亚马逊利什曼原虫的体内及体外研究。
[Abstract]:Objective: to establish an in vitro culture system of Leishmania Amazon in order to obtain a large number of flagellate for immunological and molecular biological studies. It was further proved that the antigen specificity, gene expression level and host infection characteristics of non-flagellum transformed by proflagellum were further proved. Methods: by improving the culture medium, calf serum was increased from 20% to 50%, the proflagellum was transformed into flagellum in vitro, and pure culture was carried out in vitro. And then for three different sources of flagellum (animal skin lesions, J774 macrophage infection source and proflagellum transformation): indirect immunofluorescence staining of specific monoclonal antibodies was used to analyze different sources of non-flagellum (P 鈮，

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