| Objective Construction, amplifycation and extraction of PPARγ, PPARα and PPRE expression vectors, and developing a highly sensitive screening system for PPAR ligands. Methods DNA fragments of mPPARγ and mPPARα were amplified by RT-PCR, and subcloned into pCMX expression vector, synthesized DNA of PPRE subcloned into pGL3 luciferase expression vector, and extracted the DNA of plasmids. pGL3-CMV, PPARγ and PPRE were transfected into COS-7 cells and HepG2 cells with pRL-SV40, respectively, the cells were cultured for 24h, pGL3-CMV andvarious concentration of PGZ were added into the cultured medium. After an addition 24h incubation, the luciferase activity was determined by using a Dual-Luciferase Reporter Gene Assay system. Results (1) Extracted expression vectors of PPARγ PPARα and PPRE were sequenced that no misincorporated mutations were introduced into the PCR product during amplification and in the correct reading frame, and mainly showed ringed DNA (2) pGL3- CMV significant increase in luciferase activity by about 14.9-fold (P<0 .01) in cultured COS-7 cells.TGZ stimulate luciferase activity in a concentration- dependent (10-9, 10-8, 10-7, 10-5 mol/L) manner. TGZ 10-5 stimulate luciferase activity reached the peak by about 6.1-fold compare with control (P<0.01). (3) Incubation of HepG2 cells with the concentration of TGZ 10-6, 10-5, strongly increase luciferase activity by about 3.6-fold (P<0.01) or 5.4-fold (P<0.01), respectively. Conclusion We successfully performed the construction, amplifying and extraction of plasmids of PPARy PPARa and PPRE, successfully developed a highly sensitive luciferase screening system for PPAR ligands.
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