SiRNA特异性抑制幽门螺杆菌CagA基因表达的实验研究  

Specific inhibition of the cagA gene expression in Helicobacter pylori by small interfering RNA

作　　者：严燕国[1] 詹文华[1] 赵刚[1] 马晋平[1] 蔡世荣[1] 

机构地区：[1]中山大学附属第一医院胃肠胰外科,广州510080

出　　处：《中国人兽共患病学报》2006年第4期330-333,341,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金资助项目(No.30271276)

摘　　要：目的探讨小分子干扰RNA(SiRNA)对幽门螺旋杆菌CagA基因表达的影响。方法设计5条不同序列的针对CagA基因的的小分子干扰RNA和一条非特异性的小分子干扰RNA,采用电穿孔法转入幽门螺杆菌,以半定量RT-PCR法检测穿孔前和穿孔后1h、6h、12h、24h、48h细菌CagAmRNA的表达并用Westernblot检测CagA蛋白表达。结果电穿孔后其中1条小分子干扰RNA-SiRNAⅢ作用的CagA-mRNA显著地呈一过性表达下降,6h达最低值,1h、6h、12h、24h表达水平分别为电穿孔前的36.7%、31.3%、43.5%、76.8%(P<0.05),抑制率为68.7%,Westernblot显示12h带最弱;而另1条小分子干扰RNA-SiRNAⅤ作用较弱,抑制率为23.1%。其余3条及非特异性的小分子干扰RNA未见变化。结论SiRNA可特异性抑制幽门螺杆菌CagA基因表达。To investigate the effect of the small interfering RNA （siRNA） on the expression of CagA protein in Helicobacter pylori, 5 different sequences of siRNA and another siRNA with random sequences were designed for use to target upon the cagA sequences, and they were transferred into H. pylori through electroporation. The expression of the cagA mRNA was detected by means of the semi-quantitative RT-PCR before and after 1, 6, 12, 24 and 48 hrs of electroporation. , and Western blot assay was used to detect the expression of cagA mRNA and CagA protein. The experimental results showed that the temporarly reduced expression of one siRNA ,i. e. siRNA-Ⅲ was evident after electroporation, with a maximal level after 6 hrs of electroporation. It was also demonstrated that the expression levels after 1, 6, 12, 24 hrs after electroporation were of the 36. 7%, 31.3%, 43.5 and 76.8% in comparison with that before electroporation. , with an inhibition rate of 68.7% by siRNA- Ⅲ. The expression level of CagA protein degraded at 48 hrs after electroporation as demonstrated in Western blot assay, and another siRNA, siRNA-Ⅳ also showed weak effect on the expression of cagA MRNA, with an inhibition rate of 23.1% , while the other siRNAs showed no significant effect on the expression of eagA mRNA. It is evident from the above observations that the small interfering RNA can inhibit specifically the expression of CagA protein in H. pylori.

关 键 词：幽门螺杆菌 CAGA 小分子干扰RNA 

分 类 号：R378[医药卫生—病原生物学]