应用siRNA技术抑制产肠毒素大肠杆菌LT基因表达的研究

The study of using siRNA technology to silence expression of LT gene of Enterotoxigenic Escherichia coli

目的应用小干扰RNA(siRNA)技术抑制产肠毒素大肠杆菌(ETEC)LT基因的表达。方法针对LT基因设计siRNAs序列,在ETEC培养过程中,将针对LT的siRNA、非特异性对照siRNA、阴性对照siRNA和LB培养基分别加入siRNA组(siRNA-LT1组、siRNA-LT2组)、siRNA-coa3组、siRNA-NC组和空白对照组,分3个时间点加入,每次1nmol。在首次加入siRNA后45min(A时间点)、90min(B时间点)、135min(C时间点)留取菌液,应用实时荧光定量PCR检测上述3个时间点5组LT mRNA的表达水平。应用蛋白免疫印迹法(Western blot)检测siRNA-LT1组、siRNA-LT2组和空白对照组在3个时间点的LT蛋白表达水平。结果实时荧光定量PCR结果显示,siRNA-LT1在A、B、C 3个时间点对LT mRNA表达的抑制率分别为70.9%、70.1%、72.5%,siRNA-LT2在A、B、C 3个时间点对LT mRNA表达的抑制率分别为70.1%、69.2%、70.5%,siRNALT1组、siRNA-LT2组对LT mRNA表达的抑制率与相应处理时间的siRNA-NC组、siRNA-coa3组、空白对照组比较差异均有统计学意义(P<0.05)。Western Blot结果显示,siRNA-LT1和siRNA-LT2在A、B、C 3个时间点对LT蛋白表达的抑制率分别为43.1%、18.4%、5.0%和38.2%、15.4%、30.1%。结论针对LT基因设计合成的siRNA在体外能有效地抑制LT基因的表达。

The distinctive LT siRNAs were designed according to the LT sequence .During the process of cultivation ,siRNA targeting the LT gene ,non‐specific control siRNA ,negative control siRNA and culture medium were added into siRNA group （siRNA‐LT1 group , siRNA‐LT2 group） ,siRNA‐coa3 group ,siRNA‐NC group and blank control group ,respectively ,and three times in each group （1 nmol each time） .After siRNA added at the first time ,bacteria was collected in 45 min （A） ,90 min （B） and 135 min （C） time points .The expression of mRNA in three time points （A ,B and C） were detected by real‐time fluorescence quantitative PCR .The protein level of LT in siRNA‐LT1 group ,siRNA‐LT2 group and blank control group were detected by Western blot in three time points .Results The results of real‐time fluorescence quantitative PCR showed that inhibition of siRNA‐LT1 on the expression of LT mRNA at the three time points（A ,B and C）were 70 .9% ,70 .1% ,72 .5% respectively ,and inhibition of siRNA‐LT2 on the ex‐pression of LT mRNA at the three time points（A ,B and C）were 70 .1% ,69 .2% and 70 .5% respectively .In the three time points （A ,B and C）the inhibition rate of the expression of LT mRNA in siRNA‐LT1 group and siRNA‐LT2 group were statistically lower than that in the siRNA‐NC group ,siRNA‐coa3 group and blank control group （P〈0 .05） .The results of Western blot showed that in siRNA‐LT1 group the inhibitory rate of expression of LT protein in the three time points were 43 .1% ,18 .4% and 5 .0% ,re‐spectively ;in the siRNA‐LT2 group were 38 .2% ,15 .4% and 30 .1% ,respectively .Conclusion The specific siRNA could inhibit the expression of LT gene in vitro .