大鼠siTLR4重组腺病毒载体的构建及功能鉴定

目的构建特异性针对大鼠TLR4基因的siRNA重组腺病毒载体,为进一步研究TLR4在不同疾病中的免疫调节机制奠定重要基础。方法设计并合成3对大鼠TLR4基因的siRNA序列,退火处理后定向克隆到pSES-HUS穿梭质粒中,获得pSES-HUS-siTLR4,经Pme I线性化后与pAdEasy-1骨架质粒在BJ5183细菌中进行同源重组,从而构建pAd-siTLR4质粒,通过脂质体转染,在HEK293细胞中包装形成Ad-siTLR4腺病毒颗粒,用该病毒感染PC12细胞,从基因和蛋白质水平分别检测3对siTLR4的抑制效果,同时从蛋白质水平检测TLR4下游关键分子NF-κB的表达。结果 PCR、酶切和测序均证实目的基因正确克隆到所构建的pAd-siTLR4重组腺病毒载体中;经包装获得的Ad-siTLR4病毒颗粒在PC12细胞中能够有效地抑制TLR4 mRNA和蛋白水平的表达,并显著地降低了NF-κB的表达。结论成功构建了pAd-siTLR4重组腺病毒质粒,同时包装获得了Ad-siTLR4重组腺病毒,转染PC12细胞后不仅能明显降低TLR4的表达,而且有效地抑制了TLR4通路下游关键分子NF-κB的表达。

Construction and functional identification of rat siTLR4 recombinant adenovirus vector

We aimed to construct the recombinant adenovirus vector carrying specific small interfering RNA(siRNA) targeting TLR4 gene of rat,for further research of the immunoregulatory mechanisms of TLR4 in different diseases.Three pairs of siRNA sequence for silencing rat TLR4 gene were designed,then annealed and cloned into the pSES-HUS vector in order to gain pSES-HUS-siTLR4 plasmid.After the recombinant vector was linearized by Pme I,the pAd-siTLR4 plasmid was obtained by homologous recombination between the pSES-HUS-siTLR4 and the backbone vector pAdEasy-1 in E.coli BJ5183.Afterward,the pAd-siTLR4 plasmid was transfected into HEK293 cells through Lipofectamine 2000 to package Ad-siTLR4 recombinant adenovirus.Then Ad-siTLR4 recombinant adenovirus was used to infect PC12 cells,and the levels of TLR4 mRNA and protein expression were detected by Real-time PCR and Western blotting.We also tested the changes of NF-κB expression,which is a key protein in the TLR4 signaling pathway.Real-time PCR and Western blotting indicated that TLR4 mRNA and protein levels were significantly reduced in infected PC12 cells.Meanwhile,the expression level of NF-κB was also obviously decreased.All of these suggested that the pAd-siTLR4 recombinant adenovirus vector was constructed successfully and Ad-siTLR4 recombinant adenovirus could effectively suppress TLR4 expression and obviously reduce level of NF-κB expression in PC12 cells.