沉默Toll样受体4基因表达的RNAi慢病毒载体的构建******

背景：Toll样受体4 (toll-like receptor4，TLR4)是介导内毒素/脂多糖应答的主要受体，在由内毒素诱导的炎性反应的信号通路中发挥着重要作用。 目的：构建人TLR4基因的RNA干扰慢病毒载体，并观察其对人脐静脉内皮细胞TLR4在蛋白水平的沉默效应。 方法：利用Invitrogen在线软件设计人TLR4基因shRNA序列，合成、退火形成双链寡核苷酸后克隆到线性载体pENTRTM/H1/TO的黏性末端，并进行DNA测序。得到的阳性重组子再与慢病毒载体进行重组反应，从而获得干扰TLR4基因真核表达的慢病毒载体。在脂质体的介导下将慢病毒包装辅助复合体和TLR4基因的真核表达慢病毒载体导入293FT细胞包装病毒，测定病毒滴度，感染人脐静脉内皮细胞，检验其干扰TLR4基因表达的有效性。 结果与结论：实验成功构建TLR4基因真核表达慢病毒干扰载体并获得相应的慢病毒，病毒滴度为8.7×106 U/mL。免疫印迹杂交结果表明，所获得的慢病毒感染人脐静脉内皮细胞TLR4基因在蛋白水平的表达显著降低。实验成功构建了人TLR4基因慢病毒RNA干扰表达载体，并验证了其在人脐静脉内皮细胞上的有效性。

Construction of a lentiviral vector for RNA interference targeting human toll-like receptor 4 gene

BACKGROUND: As the most significant receptor mediating endotoxin or lipopolysaccharide response, toll-like receptor 4 (TLR4) plays a key role in the signaling pathway of inflammation induced by endotoxin. OBJECTIVE: To construct a lentiviral RNAi vector that is capable of knocking down TLR4 gene in human umbilical vein endothelial cells (HUVECs). METHODS: Short hairpin RNA (shRNA) sequence targeting Human TLR4 gene was designed using the Invitrogen online designing software. After synthesis and annealing, the double-stranded oligo nucleotides (ds oligo) were cloned into pENTRTM/H1/TO vectors and the resulting entry clones were identified by sequencing. Then, a recombinant reaction was performed to transfer the RNAi cassette from the entry plasmids into pLenti4/BLOCK-iTTM-DEST vectors to create the expression clone. Recombinant lentivirus was harvested from 293FT cells contransfected with the expression plasmids and the ViraPowerTM packing Mix. HUVEC cells were infected with the recombinant lentivirus and TLR4 expression in these cells was subsequently detected by western blot. RESULTS AND CONCLUSION: Recombinant lentivirus expressing shRNA targeting the TLR4 gene was successfully constructed with the titer of 8.7×106 U/ml. Western blot result showed that the expression of TLR4 in lentivirus-infected HUVEC cells was significantly lower than that in the control cells. The lentiviral RNAi vector with the capability of knocking down TLR4 gene in HUVEC cells has been successfully constructed, providing a basis for further study on the relationship between inflammation and TLR4 gene.