弓形虫基因缺陷虫株建立方法的初步研究

Preliminary Research of Establishment Method on Toxoplasma Gondii Knockout Strain

目的:应用电穿孔技术转染Tg P24基因敲除转染质粒于弓形虫RH,探讨电穿孔技术的应用条件,以及Tg P24基因敲除质粒转染虫株在不同哺乳动物细胞中筛选的最适条件。方法:设定所需的电穿孔参数、条件,如电压,电容,脉冲次数,电击杯的大小,电转染缓冲液,电穿孔后,将弓形虫悬浮液分别转移到长有不同贴壁细胞的培养瓶中,37℃,5%CO_2(体积分数)的培养箱中培养,观察弓形虫的生长状况,并对不同电穿孔参数、条件下的弓形虫存活率进行比较。12hr后换成加有福来霉素的完全培养基中选择培养,不同时间观察虫体及细胞生长情况;在细胞中选择培养10天后,收集虫体,4℃下福来霉素处理7天,再回复到细胞内用选择培养基培养5天,收集虫体,腹腔注射昆明小鼠,大量收集弓形虫用于提取RNA进行RT-PCR。结果:优化电穿孔条件后的弓形虫存活率得到提高(P<0.05);在选择浓度为5.0μg/ml-7.5μg/ml的福来霉素培养基中,筛选虫株采用L929细胞做为宿主细胞最为适合;RT-PCR结果显示L929细胞筛选基因敲除虫株的效果较好。结论:初步确定了弓形虫电穿孔技术的应用条件,以及TgP24基因敲除质粒转染虫株在不同哺乳动物细胞中的最佳筛选条件;获得了较好的转染效率,为进一步研究弓形虫TgP24基因敲除株的生物学特性打下了良好的基础。

Objective Using electroporation technology to transfect TgP24 gene knockout plasmid into the Toxoplasma RH, Exploration and Discussion on the application of electroporation conditions,and optimum screening conditions of strains in different mammalian cells. Methods Setting the parameters and conditions for electroporation conditions,such as voltage,capacitance,pulse frequency,the size of electroporation cuvette, electroporation transfer buffer. After electroporation,Toxoplasma gondii suspension respectively was transferred to bottles cultured different adherent cell,in the incubator with 37 ℃,5% CO2 （volume fraction）,observed the growth of Toxoplasma gondii. After 12 hours,Toxoplasma gondii were cultured in culture medium added phleomycin,and observed the status of strains and cell growth in different time. collection of strains was after 10 days selective culture in 4℃ in 7-day phleomycin selection. Strains were cultured in the selective medium for 5 days. Than collect the strains were collected and injected into mice celiac. RNA of collected Toxoplasma gondii was extracted for follow-up test. Results Optimized conditions of electroporation had improved the survival rate of Toxoplasma gondii （P 〈0.05）. L929 cells were most suitable for screening knockout strains in culture medium with 5.0 μ g/ml-7.5 μ g / ml phleomycin. RT-PCR results showed that L929 cells had the good screening effect for the knockout strains. Conclusion Preliminary ascertain the application conditions of Toxoplasma electroporation technology,and the best screening conditions of TgP24 knockout plasmid transfected into RH strains cultured in different mammalian cell lines. Efficient transfection is obtained,It has laid a favourable foundation for further research on the biological characteristics of TgP24 knockout strains.