中文摘要：

目的构建含有Toll样受体4（TLR4）基因特异性RNA干扰序列的pSliencer4.1-CMV neo载体,比较其对TLR4的抑制效率。方法通过Ambion公司的在线软件设计TLR4基因（GenBank：NM138554）的RNA干扰（RNAi）序列,按siRNA设计的优化原则筛选靶序列,再选取其中部分序列进行合成,经退火、连接,构建含有TLR4基因特异性RNA干扰序列的pSliencer4.1-CMV neo载体。用脂质体SiPort XP-1分别将载体及阳性和阴性对照质粒转染血管内皮细胞系EVC-304细胞,提取细胞总RNA,进行RT-PCR反应,筛选抑制效率较高的载体。结果共设计出可供选择的序列228条,经BLAST比对后,有30条序列有较好的序列特异性,并根据其在基因中的相对位置选择9条作为siRNA靶序列。RT-PCR结果表明其中有5个载体具有较高的抑制效率,以TS4,TS6,TS8抑制效率最高,分别为89%,90%,88%,其余siRNA的抑制效率均未超过50%。抑制效果较好的5条序列在TLR4基因中的位置分别为444、1203、1389、2774、3409nt处。结论成功构建了抑制效率较高的TLR4基因特异性RNA干扰序列的pSliencer4.1-CMV neo载体,筛选出特异性抑制TLR4表达的序列,为TLR4信号转导的研究奠定了基础。

英文摘要：

Objective To construct the TLR4 gene-specific recombinant vectors pSliencer 4. 1-CMV neo, and compare the silence effects of these small interference RNAs to TLR4. Methods Specific small interference RNAs（siRNA）were designed by the Ambion＇s online software and screened with the optimize principles. The sequences were then synthesized, annealed and recombined into the pSlieneer 4. 1-CMV neo expression vector to construct the siRNA expression vectors specific to TLR4 gene. The recombinant vectors, negative control and positive control were then transfected into the vascular endothelial cell lines （EVC-304 cells） by SiPort XP-1, respectively. The total mRNA of the EVC-304 cells was then extracted and the TLR4 expression level was detected by RT-PCR. The vector with the highest inhibition rate was screened. Results Two hundred and twenty-eight sequences were found by retrieval with the online software, while only 30 sequences accorded with the optimize principles. Nine out of the 30 sequences were selected and constructed into the pSliencer 4. 1- CMV nco expression vector. RT-PCR showed that 5 sequences, namely TS1, TS2, TS4, TS6 and TS8, had excellent silence effect to TLR4. Three sequences, namely TS4, TS6 and TSS, had the highest silence effect to TLR4 mRNA expression, the inhibition ratios were 89%, 90% and 88%, respectively. Those 5 siRNA sequences with excellent silence effect to TLR4 were located at 444nt, 1 203nt, 1 389nt, 2 774nt and 3 409nt of TLR4 gene, respectively. Conclusions Recombiant expression vectors have been successfully constructed, and the most excellent sequences have been selected. It is beneficial in laying the foundation for the further study of TLR4 signaling pathways.