Zhang Yun, Gong Min, Bi Yang, Jiang Wei, Yu Qin, Li Ting-yu, Chen Jie
Children's Hospital of Chongqing Medical University, Chongqing, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2012 Feb;28(2):144-7.
To construct the recombinant adenovirus vector containing specific small interfering RNA (siRNA) targeting rat TLR2 gene and identify its function in PC12 cells.
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.
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.
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.
构建靶向大鼠TLR2基因的特异性小干扰RNA（siRNA）重组腺病毒载体，并鉴定其在PC12细胞中的功能。
体外退火3对用于沉默大鼠TLR2的双链DNA片段，然后定向克隆到pSES-HUS载体中构建pSES-HUS-siTLR2质粒。之后，用PmeI将正确的重组体线性化，随后与骨架载体pAdEasy-1在大肠杆菌BJ5183中共转化以构建pAd-siTLR2质粒，然后通过脂质体转染到HEK293细胞系中包装腺病毒。用腺病毒Ad-siTLR2感染PC12细胞，通过实时荧光定量PCR和蛋白质免疫印迹法检测siRNA的抑制作用。
通过质粒PCR和基因测序，验证了siTLR2靶基因正确克隆到腺病毒载体中。通过实时荧光定量PCR和蛋白质免疫印迹法，发现用腺病毒Ad-siTLR2感染的PC12细胞中TLR2表达显著降低。
成功构建了含大鼠siTLR2基因的重组腺病毒载体，并在HEK293细胞系中包装了腺病毒，其可有效降低PC12细胞中TLR2的表达，便于研究TLR2在不同疾病中的免疫调节机制。
