大鼠Toll样受体2基因siRNA重组腺病毒载体的构建及鉴定

Construction and identification of recombinant adenovirus vector containing siRNA for rat TLR2 gene

目的:构建特异性抑制大鼠TLR2基因的重组腺病毒载体,并在大鼠嗜铬细胞瘤PC12细胞中进行功能鉴定。方法:体外合成3对大鼠siTLR2的双链DNA序列,经退火定向克隆到穿梭质粒pSES-HUS中获得pSES-HUS-siTLR2质粒,Pme I线性化后在BJ5183细菌中与pAdEasy-1骨架质粒进行同源重组获得pAd-siTLR2质粒,脂质体转染HEK293细胞,包装获得Ad-siTLR2腺病毒颗粒,继而感染PC12细胞,通过Real-time PCR和Western blot方法检测3对siRNA对TLR2基因的抑制效率。结果:PCR凝胶电泳和测序结果均证实目的基因正确克隆到腺病毒载体中;Real-time PCR和Western blot结果显示:携带siTLR2的病毒颗粒能够在mRNA和蛋白水平有效抑制PC12细胞中TLR2的表达。结论:成功构建了Ad-siTLR2重组腺病毒载体,并在HEK293细胞中包装成重组腺病毒,转染PC12细胞后能有效抑制TLR2基因的表达,为进一步研究TLR2在不同疾病中的免疫调节机制奠定重要基础。

AIM： To construct the recombinant adenovirus vector containing specific small interfering RNA（siRNA） targeting rat TLR2 gene and identify its function in PC12 cells.METHODS： Three pairs of double-stranded DNA fragments for silencing rat TLR2 were annealed in vitro,then directional cloned into the pSES-HUS vector to construct pSES-HUS-siTLR2 plasmid.Afterward,the correct recombinant was linearized by PmeI,following co-transformation with the backbone vector pAdEasy-1 in E.coli BJ5183 to construct pAd-siTLR2 plasmid,and then transfected into HEK293 cell line via Lipofectamine to package the adenovirus.PC12 cells were infected with the adenovirus Ad-siTLR2,and inhibition of siRNA was detected with Real-time PCR and Western blotting.RESULTS： Using plasmid PCR and gene sequencing,the siTLR2 target gene was verified to be correctly cloned in the adenovirus vector.Trough Real-time PCR and Western blotting,TLR2 expression was significantly decreased in the PC12 cells which was infected with the adenovirus Ad-siTLR2.CONCLUSION： Successfully constructed the recombinant adenovirus vector containing rat siTLR2 gene and packaged the adenovirus in HEK293 cell line,which could effectively reduce TLR2 expression in the PC12 cells to facilitate the study of the immunoregulation mechanisms of TLR2 in different diseases.