活体成像技术在实验脑型疟中的应用研究

发布时间：2018-03-22 16:27

本文选题：伯氏疟原虫ANKA株　切入点：荧光素酶　出处：《中国病原生物学杂志》2017年02期 　论文类型：期刊论文

【摘要】：目的构建表达Luciferase的伯氏疟原虫ANKA株(Plasmodium berghei ANKA),感染昆明小鼠并于实验脑型疟发生时对小鼠进行活体成像,建立实验脑型疟活体成像平台,为脑型疟发病机理及疟疾疫苗和药物评价研究奠定基础。方法大量制备重组质粒pl0027并酶切使其线性化。复苏冻存原虫并于体外培养富集成熟裂殖体。收集成熟裂殖体与线性化的质粒混匀后进行电转化,对构建的虫株进行药物筛选;提取疟原虫基因组,进行PCR鉴定。构建的伯氏疟原虫ANKA-Luciferase疟原虫采用常规方法感染昆明小鼠,当小鼠于感染6d后出现偏瘫、昏迷及视网膜病变等典型脑型疟症状时,采用活体成像技术观察疟原虫增殖及分布情况;解剖分离感染小鼠各脏器组织并成像,观察疟原虫在各器官组织粘附情况。结果 PCR检测两端插入序列及Luciferase序列均能获得预期片段,提示重组质粒成功整合入伯氏疟原虫ANKA基因组内。活体成像观察感染小鼠在小鼠脑型疟发生时体内原虫水平较高,且随血流呈全身性分布;器官成像观察原虫在小鼠各重要脏器均有粘附,特别是脑部聚集了大量的感染疟原虫的红细胞。结论成功构建了伯氏疟原虫ANKA-Luciferase疟原虫,并利用此原虫感染小鼠建立了实验脑型疟活体成像平台。
[Abstract]:Objective to construct ANKA strain of Plasmodium berghei Luciferase expression (Plasmodium berghei ANKA), for in vivo imaging of mice and Kunming mice infected on experimental cerebral malaria occurs, the establishment of experimental cerebral malaria in vivo imaging platform, lay the foundation for the evaluation of cerebral malaria pathogenesis and malaria vaccines and drugs. Methods preparation of recombinant plasmid pl0027 and restriction enzyme to linearize the cryopreservation and recovery of protozoa. In vitro enrichment mature schizont. Mature schizonts collected and split plasmid linearized mixed electric conversion, drug screening of strains; extracting the Plasmodium falciparum genome, identified by PCR. The construction of Plasmodium berghei ANKA-Luciferase using conventional methods of Kunming mice infected mice after infection with 6D, when the emergence of hemiplegia, coma and retinopathy and other typical symptoms of cerebral malaria, using in vivo imaging observation of Plasmodium. Reproduction and distribution; dissection of infected mice in each organ and imaging observation of Plasmodium in different organs tissue adhesion. The result of PCR detection and Luciferase sequences were inserted into both ends can be expected, suggesting that the recombinant plasmid was successfully integrated into the genome of Plasmodium berghei ANKA. Imaging observation of infected mice in mouse cerebral malaria when the level of the body and blood flow with high parasitemia, a systemic organ distribution; imaging observation of protozoa in mouse organs have important adhesion, especially in the brain has gathered a large number of infected red blood cells. Conclusion the successful construction of Plasmodium berghei ANKA-Luciferase, and using the parasitemia in infected mice established experimental cerebral malaria in vivo imaging platform.

【作者单位】：第三军医大学基础医学部病原生物学教研室;
【基金】：国家自然科学基金项目(No.81301456) 重庆市自然科学基金项目(No.cstc2012jjA10048)
【分类号】：R531.3

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