Objective:To explore the expression and effect of the recombinant vector of human PAX8/ PPARγ fusion gene (PPFP) in human thyroid follicular epithelial cell Nthy ori 3-1.
Methods:The PAX8 gene was isolated and amplified by PCR technique from PAX8- pOTB7 plasmid. The PPARγ gene was isolated and amplified by PCR technique from PPARγ- pCMV-SPORT6 plasmid. Then the two genes were directionally united and this united gene was subcloned into the eukaryotic expression plasmid of pEGFP-C1 vector to generate the eukaryotic expression recombinant vector, pEGFP-C1-PAX8/ PPARγ.The correct PPFP gene was confirmed by PCR, edoenzyme digestion, sequencing, analysis, and contrast. After the co-transfection of pEGFP-C1-PAX8/ PPARγ and lipofectamine 2000 into human thyroid follicular epithelial cell Nthy ori 3-1,the expressions of PPFP fusion gene on the levels of mRNA and protein were detected by RT-PCR and Western blot.
Results:The correct eukaryotic expression recombinant vector, pEGFP-C1-PAX8/ PPARγ,was confirmed by verification.After the pEGFP-C1-PAX8/ PPARγ and Lipofectamine 2000 were transfected into Nthy ori 3-1 cells, the expressions of PPFP fusion gene on the levels of mRNA and protein were successfully detected.
Conclusions:The PPFP fusion gene and the eukaryotic expression recombinant vector pEGFP-C1-PAX8/ PPARγ have been constructed successfully. After this recombinant vector is transfected into Nthy ori 3-1 cells,it could successfully express PPFP protein，and provides an experimental basis for further exploring the molecular mechanisms of PPFP′s tumorigenesis.