The Study Of Using SiRNA Technology To Silence Expression Of LT Gene Of Enterotoxigenic Escherichia Coli

Objective To inhibit the expression of LT gene of Enterotoxigenic Escherichia coli by siRNA technology.Methods The distictnive LT siRNAs was designed according to the LT sequence and were synthesized in invtro by chemical synthesis method. We drawed the time-OD600curve of ETEC to judge the logarithmic phase of ETEC. At the same time, we create the equation of OD600-viable number to judge the bacrerial count in the liquid. During the process of cultivation, we added siRNA targeting the LT gene, non-specific control siRNA, negative control siRNA and culture medium to siRNA group, non-specific control (siRNA-coa3) group, negative control siRNA (siRNA-NC) group and blank control (Blank) group, respectively. We added three times in each group,1nmol each time. After the first time to add siRNA, we collected bacteria in60min (group A),90min (group B) and135min (group C) time points. The expression of mRNA in three time points (group A,45min; group B,90min; group C,135min) were detected by real-time fluorescence quantitative PCR. In every time point, we detected5groups (siRNA-LT1, siRNA-LT2, siRNA-coa3, siRNA-NC and Blank). The protein level of LT in three groups (siRNA-LT1, siRNA-LT2and Blank) were detected by Western Blot in three time point.Results According to the time-OD600curve of ETEC, we considered that OD600value between0.192and0.791is bacteria logarithmic phase. The equation of OD600-viable number:y=0.23+1.31x (R2=0.96), OD600value (x), viable number (y×109)/ml. The results of real-time fluorescence quantitative PCR showed in group A, B and C that the expression of the LT mRNA in siRNA-LT1group and siRNA-LT2group were lower than the controlled group (P<0.05). The results of Western Blot showed in siRNA-LT1group and siRNA-LT2group that the expression of LT protein in the three point were reduced, comparing to the Blank group. The inhibitory rate was43%peaking at45min in siRNA-LT1group, with the ratio of molecules of siRNA-LT1and bacrerial count was at4.76X105:1.Conclusions The specific siRNA could inhibit the expression of LT gene in vit-ro, we get two pairs of effective siRNA sequences.