伯氏疟原虫类巨噬细胞移动抑制因子的表达、纯化及其在红内期表达的检测

发布时间：2018-01-18 16:20

  本文关键词：伯氏疟原虫类巨噬细胞移动抑制因子的表达、纯化及其在红内期表达的检测　出处：《第四军医大学》2006年硕士论文　论文类型：学位论文

  更多相关文章： 伯氏疟原虫 巨噬细胞移动抑制因子 基因表达 多克隆抗体 免疫印迹

【摘要】：目的：疟疾是世界上对人类危害最严重的传染病之一。目前对疟疾的防治效果不理想，，其原因在于对疟原虫的生物学特性及其与宿主之间的相互作用缺乏了解。疟原虫基因组计划的实施，使人们发现大量未知功能的基因。对未知功能基因的研究不仅给深入了解疟原虫生物学特性提供巨大的帮助，而且为寻找新的药物作用靶点和疫苗的研制提供新的思路。巨噬细胞移动抑制因子(MIF)是较早发现的广泛存在于人及其他哺乳动物的细胞因子，可广泛参与哺乳动物的多种生物反应，特别是免疫功能的调节。疟原虫也具有MIF的同源基因。对疟原虫类MIF的研究可能会给深入了解疟原虫的生物学特性提供帮助。为研究疟原虫MIF生物学功能，本研究拟对伯氏疟原虫类巨噬细胞移动抑制因子(PbMIF)进行克隆和表达，获取纯化的可溶性蛋白，制备小鼠抗PbMIF多克隆抗体，并检测PbMIF在伯氏疟原虫红内期的表达。 方法：根据GenBank中PbMIF mRNA的预测序列，设计特异引物，采用RT-PCR方法从伯氏疟原虫ANKA株红内期RNA扩增获得PbMIF基因。将PbMIF基因与T载体连接并测序，利用NCBI中Blast程序分析测序结果。阳性T／A克隆质粒经BamH Ⅰ和Xho Ⅰ双酶切后，将目的片段克隆至原核表达载体pET28a，并转化大肠埃希菌BL21(DE3)，收集经IPTG诱导表达的
[Abstract]:Objective: malaria is one of the most serious infectious diseases in the world. This is due to the lack of understanding of the biological characteristics of Plasmodium and its interaction with the host, and the implementation of the Plasmodium Genome Project. The study of unknown functional genes not only provides a great help for further understanding the biological characteristics of Plasmodium falciparum. And it provides a new way to find new drug target and vaccine. Macrophage migration inhibitory factor (MIF) is widely found in human and other mammalian cytokines. It can be widely involved in a variety of mammalian biological reactions. In particular, the regulation of immune function. Plasmodium also has the homologous gene of MIF. The study on MIF of Plasmodium may be helpful to understand the biological characteristics of Plasmodium, and to study MIF biology of Plasmodium. Learning function. In this study, Plasmodium berghei macrophage migration inhibitor PbMIF was cloned and expressed to obtain the purified soluble protein and to prepare mouse anti-PbMIF polyclonal antibody. The expression of PbMIF in erythrocyte of Plasmodium berghei was detected. Methods: according to the predicted sequence of PbMIF mRNA in GenBank, specific primers were designed. The PbMIF gene was amplified by RT-PCR from the erythroid RNA of Plasmodium berghei ANKA strain. The PbMIF gene was ligated with T vector and sequenced. The positive T / A clone plasmid was digested by BamH 鈪

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