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

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.