利用Lifeact法检测恶性疟原虫F-actin的研究

Development of lifeact method for detecting F-actin of plasmodium falciparum

关键词:  恶性疟原虫  纤维状肌动蛋白  免疫共沉淀

Key words:  plasmodium falciparum  F-actin  immunoprecipitation

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 develop a green fluorescent protein fusion Lifeact peptide method for detecting F-actin of Plasmodium falciparum. Methods The recombinant plasmid Lifeact-GFP / p LN was constructed and transfected into P. falciparum 3D7 standard strain. The stable transfectanted cells carrying Lifeact-GFP / p LN were obtained by Blasticidin S hydrochloride（ BSD） drug screening,and the expression of Lifeact-GFP fusion protein in transfectent cells was confirmed with Western blotting and living-cell fluorescence method. The interaction of Lifeact-GFP with F-actin was detected by Co-immunoprecipitation tests（ Co-IP）. Results Lifeact-GFP transgenic strains were successfully acquired, which were confirmed by Western Blotting and fluorescence detection. Co-immunoprecipitation show ed that Lifeact was not bound to F-actin of P. Falciparum directly.Conclusion The results show that Lifeact peptides can specifically bind to F-actin in yeast cells,however,it cannot identify F-actin of plasmodium falciparum.