胞浆表达绿色荧光蛋白的伯氏疟原虫的构建和筛选

目的:为了追踪疟原虫发育及侵染的情况，构建及筛选胞浆稳定表达绿色荧光蛋白（GFP）的伯氏疟原虫。方法:构建含有伯氏疟原虫230p基因和GFP基因的重组质粒pL0035-GFP，质粒线性化后通过电转染转入野生型伯氏疟原虫；基于双交换同源重组原理，利用乙胺嘧啶筛选获得在230p基因处插入GFP的重组疟原虫；有限稀释法筛选表达GFP的单克隆重组伯氏疟原虫；PCR鉴定重组疟原虫基因型，荧光显微镜和流式细胞术检测GFP表达情况。结果:PCR及DNA测序结果表明GFP基因成功整合到伯氏疟原虫基因230p；荧光显微镜可观察到重组疟原虫胞浆呈绿色荧光，流式细胞术检测到绿色荧光信号。结论:成功构建胞浆表达绿色荧光蛋白的伯氏疟原虫。

Construction of recombinant Plasmodium berghei expressing green fluorescent protein in cytoplasm

Objective: To construct a recombinant Plasmodium berghei(P. berghei) strain stably expressing green fluorescent protein (GFP) in cytoplasm to track the development and infection of Plasmodium. Methods: The recombinant plasmid pL0035-GFP containing P. berghei 230p gene and GFP gene sequences was constructed. The plasmid was linearized and transfected into wild-type P. berghei by electroporation. Based on the principle of double-crossover homologous recombination, the recombinant P. berghei was screened by pyrimethamine treatment. The single clone of the recombinant Plasmodium falciparum expressing GFP at 230p locus was selected by limiting dilution method. The genotype of recombinant parasites was identified by PCR and DNA sequencing, and the expression of GFP was detected by flow cytometry and fluorescence microscopy. Results: PCR and DNA sequencing results showed that the GFP gene was successfully integrated into the P. berghei 230p gene locus. Fluorescence microscopy showed that the recombinant parasites expressed green fluorescence in cytoplasm, and green fluorescence signal was also detected by flow cytometry. Conclusion: We successfully construct the recombinant P. berghei expressing green fluorescent protein in cytoplasm.