摘要
目的探讨小干扰RNA(siRNA)在弓形虫体内的对目的基因的表达调节作用。方法以弓形虫嘌呤代谢过程中的关键酶——次黄嘌呤、黄嘌呤、鸟嘌呤磷酸核糖转移酶(HXGPRT)编码基因为靶标,合成3对siRNA,以电穿孔的方式将不同的浓度的siRNA转染弓形虫。采用荧光定量PCR及[3H]-次黄嘌呤摄入量分别检测弓形虫HXGPRT mRNA及酶合成量。结果在针对HXGPRT编码基因设计的3对21nt的siRNA中,有1对(siRNA415)能明显降低HXGPRT mRNA水平及酶合成量。在转染后24h,4μmol/LsiRNA415电转弓形虫的HXGPRT mRNA水平及[3H]-次黄嘌呤摄入量分别降为模拟电转弓形虫的0.36±0.04及0.51±0.03倍(P<0.05)。结论21nt的siRNA在弓形虫体内能有效抑制目的基因的表达。siRNA在弓形虫体内的应用将为阐明未知基因的功能和抗弓形虫药物的筛选提供有力的工具。
Objective To elucidate whether siRNA can effectively down-regulate the expression of target genes in T. gondii. Methods Hypoxanthine-xanthine-guanine phosphoribosyltransferase （HXGPRT） was one of the most important enzymes in the T. gondii purine salvage pathway. Three 21 nt HXGPRT siRNAs were designed and electroporated into the parasites with a concentration varying from 1 μmol/L to 4 μmol/L. The mRNA level and enzyme activity of HXGPRT in the electroporated parasites were determined by real-time quantitative PCR and [ ^3H ]-count, respectively. Results In the three designed HXGPRT 21 nt siRNAs, only one siRNA, namely siRNA415 worked effectively in T. gondii. At 24 h post-infection, the relative value of HXGPRT mRNA levels and HXGPRT activity decreased to 0. 36± 0. 04 and 0. 51 ±0. 03 （ P 〈 0. 05 ）, respectively. Conclusion The data show that siRNAs transfected into cells can efficiently regulate gene expression in T. gondii. The application of siRNA in interrupting gene expression in T. gondii provides a useful way for elucidating gene function as a step toward development of anti-toxoplasmasis vaccines and therapeutic reagents.
出处
《安徽医科大学学报》
CAS
北大核心
2008年第4期376-380,共5页
Acta Universitatis Medicinalis Anhui
基金
安徽省教育厅高校自然科学基金项目(编号:KJ2008B291)
