蚊期和红内期持续表达绿色荧光蛋白的重组约氏疟原虫

目的 构建蚊期、红内期持续表达绿色荧光蛋白（GFP）的约氏疟原虫BY265株。 方法 用SacⅡ酶酶切含有伯氏疟原虫ssu-rrna基因和GFP基因的重组质粒pl0017使之线性化,用电转化的方法将该重组质粒转化入红内期约氏疟原虫BY265株,获BY265-EGFP重组疟原虫,尾静脉注射感染昆明小鼠,24～30 h后用乙胺嘧啶饲喂小鼠5～6 d,鼠尾静脉采血涂片观察原虫感染率。以疟原虫基因组DNA为模板,PCR鉴定转染重组质粒pl0017的约氏疟原虫。斯氏按蚊叮咬感染BY265-EGFP重组疟原虫的小鼠,按蚊血餐后第7天和第16天解剖蚊胃和唾液腺,观察疟原虫能否在蚊体内正常发育。 结果 荧光显微镜观察到呈绿色荧光的红内期各期形态正常的约氏疟原虫。PCR检测结果表明,GFP和ssu-rrna基因已成功整合到约氏疟原虫基因组中。按蚊感染实验证实重组BY265株能在蚊体内正常发育。 结论 构建了蚊期、红内期持续表达绿色荧光蛋白的约氏疟原虫。

Recombinant Plasmodium yoelii Expressing Green Fluorescent Protein in Erythrocytic and Mosquito Stages

Objective To generate recombinant Plasmodium yoelii BY265 strain which can express green fluorescent protein （GFP） in erythrocytic and mosquito stages. Methods Recombinant plasmid containing P. berghei ssu-rrna and GFP genes were linearized by SacⅡ. The linearized plasmid was introduced into the erythrocytic stage of P. yoelii by electroporation. Kunming mice were infected with the recombinant parasites. After 24-30 hours post-infection, mice were treated with 70 μg/ml of pyrimethamine intraperitoneally for 5-6 d, and tail vein blood was then collected to make smear for parasite count. The parasites were examined by PCR. Anopheles stephensi mosquitoes were used to bite mice infected with the recombinant parasite. At the 7th and 16th day after the bite, oocyst development in mosquitoes was observed by fluorescence microscopy. Results The recombinant parasites expressed GFP in erythrocytic stage, and the GFP and ssu-rrna genes were amplified by PCR in the recombinant parasites. The mosquito infection experiment showed a normal development of the recombinant parasites. Conclusion Transgenic P. yoelii BY265 strain has been established with stable expression of GFP in both erythrocytic and mosquito stages.