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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。
Objective To construct Plasmodium berghei ANKA expressing Plasmodium berghei ANKA expressing Luciferase and to infect Kunming mice and in vivo imaging of mice during the experimental cerebral malaria and to establish an experimental platform for the imaging of brain malaria in vivo. Mechanism and evaluation of malaria vaccine and drug laying the foundation. Methods A large number of recombinant plasmid pl0027 was prepared and digested to linearize. Recovery of frozen parasite and in vitro enrichment of mature schizonts. Mature schizonts and linearized plasmids were collected and electrotransformed. The constructed strains were screened for drug selection. The genomic DNA of Plasmodium was extracted and identified by PCR. The Plasmodium berghei ANKA-Luciferase constructed by Plasmodium berghei infected Kunming mice by conventional methods. When the mice developed typical cerebral malaria symptoms such as hemiplegia, coma and retinopathy after 6 days of infection, live-imaging technique was used to observe the proliferation and Distribution; anatomy and isolation of infected organs and imaging mice to observe the malaria parasite in various organs and tissues adhesion. Results PCR detection of both ends of the insert and Luciferase sequence can obtain the expected fragment, suggesting that the recombinant plasmid was successfully integrated into the P. berghei ANKA genome. In vivo imaging showed that protozoal levels of infected mice were higher in mice with cerebral malaria and showed systemic distribution with blood flow. Organ imaging showed that protozoa had adhesion in all important organs of mice, especially in brain A large number of red blood cells infected with malaria parasites. Conclusion Plasmodium berghei ANKA-Luciferase was successfully constructed and used to infect mice with this protozoal.