方法：从含有PAX8基因和PPARγ基因的质粒克隆模板PAX8-pOTB7及PPARγ-pCMV-SPORT6中，利用PCR方法调取目的基因PAX8和PPARγ，将PAX8和PPARγ定向连接后克隆到pEGFP-C1载体上，构建融合基因的真核表达质粒pEGFP-C1-PAX8/PPARγ，在大肠杆菌E.coli DH5α中转化并提取质粒,通过PCR和测序、分析比对验证PPFP融合基因后，将真核表达载体pEGFP-C1-PAX8/PPARγ的质粒用脂质体包合并转染人正常甲状腺上皮细胞Nthy ori 3-1,通过RT-PCR和Western blot鉴定PPFP融合基因于靶细胞内在mRNA和蛋白水平上的表达。
结果：构建的质粒通过鉴定证实正确;该质粒转染人正常甲状腺上皮细胞Nthy ori 3-1后，经验证显示PPFP融合基因在mRNA和蛋白水平上顺利表达。
结论：成功克隆了PPFP融合基因并构建其重组质粒pEGFP-C1-PAX8/PPARγ；该质粒转染人正常甲状腺上皮细胞Nthy ori 3-1后可顺利表达PPFP蛋白，为进一步研究PPFP基因致瘤作用的分子机制提供了实验基础。
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.
刘剑鸣, 王志明, 李新营, 李劲东, 李萃, 段朝军, 汤参娥.人PAX8与PPARγ的融合基因（PPFP）的克隆及其重组质粒的构建[J].中国普通外科杂志,2009,18(11):1149-1155.