伯氏疟原虫电穿孔转染方案的优化  

Optimization of electroporation protocol for DNA transfection of Plasmodium berghei

作　　者：杜靖 贝祝春 王红 徐力昆 王保刚 宋亚彬 张东娜 窦媛媛 王洪权 DU Jing;BEI Zhu-chun;WANG Hong;XU Li-kun;WANG Bao-gang;SONG Ya-bin;ZHANG Dong-na;DOU Yuan-yuan;WANG Hong-quan(Institute of Microbiology and Epidemiology,Academy of Military Medical Sciences,Academy of Military Sciences, Beijing 100071,China)

机构地区：[1]军事科学院军事医学研究院微生物流行病研究所,北京100071

出　　处：《军事医学》2018年第12期949-954,共6页Military Medical Sciences

基　　金：国家重点实验室研究课题资助项目(SKLPBS1505)

摘　　要：目的通过在不同电转染方案下对电转染外源DNA到伯氏疟原虫效率进行对比,筛选优化出最佳的电转染方案。方法以mKate2为报告基因构建环状质粒p L0035-mKate2-3UTR,饲养昆明小鼠在体培养伯氏疟原虫(Plasmodium berghei),达到相应寄生率后体外同步化培养疟原虫,Nycodenz梯度离心纯化疟原虫成熟裂殖体,利用Lonza公司Nucleofector核转染系统对伯氏疟原虫进行电转染,采用流式细胞仪分析电转染效率,并提取昆明小鼠全血DNA作为模板进行PCR扩增,鉴定转染结果。结果经转染后流式细胞仪分析和血涂片吉姆萨染色镜下观察,Lonza Nucleofector 4D核转染系统电转染程序FI115与FP167均可使转染效率达到10^(-3),FP167下转染10μg环状质粒效率接近10^(-2)。在相同电转染条件下,外源DNA越多,转染效率越高。结论优化得到伯氏疟原虫电转染较好方案,为后续疟原虫药物、疫苗及生物学相关研究提供了可靠、高效的转染方案。Objective To optimize the optimal electrotransfection protocol by comparing the efficiency of electroporation of exogenous DNA to Plasmodium berghei under different electroporation conditions. Methods Firstly,mKate2 was used as a reporter gene to construct a circular plasmid pmkate2-3 UTR. P. berghei was inoculated on KM mice and underwent synchronized culture in vitro. The Nycodenz gradient centrifugation was used to purify the mature schizonts. The most advanced LONZA Nucleofector nuclear transfection system was used for the electrotransfection of P. berghei. Finally,the efficiency of electrotransfection was analyzed by flow cytometry and the whole blood DNA of infected mice was extracted as a template for PCR amplification and identification of transfection results. Results Through analysis of flow cytometry and observation of blood smear after transfection,the electrotransfection program FI115 and FP167 of Lonza Nucleofector 4 D nuclear transfection system could achieve a transfection efficiency of 10^-3. Furthermore,the transfection efficiency under program FP167 with circular plasmid of 10 μg was close to 10^-2. Under the same conditions of electrotransfection,the more the exogenous DNA,the higher the efficiency of transfection. Conclusion A reliable and efficient electroporation protocol of P. berghei has been optimized and established,which will facilitate research on potential antimalarial drugs,vaccines and functions of Plasmodium gene.

关 键 词：伯氏疟原虫 转染效率 电转染 电穿孔 流式细胞仪 mKate2 

分 类 号：R382.31[医药卫生—医学寄生虫学]