【摘要】 目的建立弓形虫SAG3基因RNAi体系,为近一步研究SAG3的功能奠定基础。方法以弓形虫SAG3基因3′-UTR区及CDS区为靶目标体外转录合成dsRNAs,通过电转法化将其分别导入弓形虫速殖子体内,用转染后的弓形虫感染SMMC7721细胞,半定量RT-PCR分析鉴定RNAi效果。结果分别于转染12、24和48h后,RT-PCR检测SAG3基因mRNA,显示dsRNA转染组SAG3mRNA水平明显低于未转染dsRNA的空白对照组和阴性对照组。其中以电压0.4kV,时间延迟为9ms的3′UTRdsRNA转染组效果最好。转染12h后,即有较明显的干扰效果。转染24h后,干扰效果最为显著。结论筛选出了可以有效抑制弓形虫SAG3基因的dsRNA序列,初步确定了RNAi发生及持续时间,为进一步研究SAG3基因功能奠定了基础。更多还原

【Abstract】 Objective To establish an RNAi system for the gene SAG3 gene in Toxoplasma gondii in order to further study its potential function.Methods Two double-stranded RNA(dsRNA) sequences corresponding to the 3’-UTR and CDS regions of the SAG3 gene were obtained using an in vitro transcription technique.The dsRNA sequences were transfected into tachyzoites of T.gondii by electroporation.The transfected parasites were cultivated in SMMC7721 cells.The effect of RNAi was detected by RT-PCR.Results To determine the effectiveness of RNAi,RT-PCR was used to detect SAG3 mRNA 12 h,24 h,and 48 h after transfection.Results demonstrated that the mRNA levels of SAG3 in the dsRNA-transfected group were significantly lower than those in the non-transfected blank and negative control groups.With a voltage of 0.4 kV and pulse time of 6-9 ms,the best moderating effect was obtained in the 3’UTR dsRNA transfection group.An obvious moderating effect was observed 12 h after transfection and reached its maximum 24 h after transfection.Conclusion dsRNA sequences that effectively inhibited the SAG3 gene were obtained.The timing and duration of RNAi were determined.An effective RNAi system for the SAG3 gene was established,laying a foundation for further research into the gene’s function.RNAi were determined.An effective RNAi system of SAG3 gene was established,laying a foundation for the further research of SAG3 function.更多还原