利用Lifeact法检测恶性疟原虫F-actin的研究

发布时间：2018-05-02 20:55

本文选题：恶性疟原虫 + 纤维状肌动蛋白　；参考：《同济大学学报(医学版)》2014年06期

【摘要】：目的通过构建游离表达绿色荧光蛋白融合Lifeact多肽(Lifeact-GFP)的转基因虫株,在不影响恶性疟原虫actin动态平衡的前提下特异标识纤维状肌动蛋白(F-actin),为研究F-actin在恶性疟原虫中的特性及其对毒力基因的转录调控作用打下基础。方法构建重组转染质粒Lifeact-GFP/p LN,经电转化方法将质粒转入恶性疟原虫3D7标准虫株中,BSD药物筛选获得携带游离Lifeact-GFP/p LN质粒的转基因虫株,并经免疫印迹(Western Blot)和活细胞荧光检测,证实Lifeact-GFP融合基因的细胞内表达,以期通过免疫共沉淀实验(Co-IP)检测Lifeact与恶性疟原虫F-actin的相互作用。结果成功获得了恶性疟原虫Lifeact-GFP转基因虫株,并通过W estern Blot和荧光检测证实Lifeact-GFP成功获得表达;但是,免疫共沉淀实验结果初步显示,Lifeact不能直接结合恶性疟原虫F-actin。结论虽然Lifeact多肽能在酵母等细胞中特异结合F-actin,但是不能直接识别恶性疟原虫F-actin。
[Abstract]:Objective to identify fibrous actin (F-actin) without affecting the dynamic balance of Plasmodium falciparum actin without affecting the dynamic balance of Plasmodium falciparum actin, and to lay a foundation for studying the specificity of F-actin in the Plasmodium falciparum and its regulatory role in the transcription of virulence genes, by constructing a transgenic insect strain expressing the free green fluorescent protein (GFP) fusion peptide (Lifeact-GFP). Methods the recombinant plasmid Lifeact-GFP/p LN was constructed, and the plasmid was transferred into the 3D7 standard strain of Plasmodium falciparum. BSD drugs were screened to obtain the transgenic plants carrying free Lifeact-GFP/p LN plasmids. The intracellular expression of the Lifeact-GFP fusion gene was confirmed by immunoblotting (Western Blot) and living cell fluorescence detection. The interaction between Lifeact and Plasmodium falciparum F-actin was detected by immunoprecipitation assay (Co-IP). Results were successfully obtained from Lifeact-GFP transgenic insect strains of Plasmodium falciparum, and the expression of Lifeact-GFP was confirmed by W estern Blot and fluorescence detection. However, the results of the immuno sedimentation experiment showed that Lifeact could not be directly combined with evil. Plasmodium falciparum F-actin. conclusion although Lifeact peptide can specifically bind F-actin in yeast cells, it can not directly identify Plasmodium falciparum F-actin..

【作者单位】：同济大学医学院;
【分类号】：R382.31

【参考文献】