小鼠增强型绿色荧光蛋白-过氧化物酶体增长因子活化受体γ2融合表达重组腺病毒的构建及表达

Objective To construct mouse enhanced green fluorecence protein（EGFP）-peroxisome proliferator - activated receptor（PPAR）γ2, and to detect EGFP-PPARγ2 expression in infected mouse bone marrow mesenchy - mal stem cells（BMSC）. Methods Cut the fragment of PPARγ2 from the expression plasmid pcDNA flag PPARγ2, then cloned the gene fragment into pEGFP-C1 and pEGFP-N1 vector. Subsequently, subclone the fragment EGFPPPARγ2 from pEGFP-C1-PPARγ2 into the shuttle plasmid DC315. HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid DC315-EGFP-PPARγ2 and large adenovirus helper plasmid pBHGlox△E1, 3Cre in mediation of liposome. The obtained replication-defective recombinant adenovirus Ad-EGFP-PPARγ2 was confirmed. Then it was propagated in HEK293 cells. After the BMSC were transfected for 72 h, adipogenic differentiation was demonstrated. Results HEK293 cells were transfected with the pEGFP-C1-PPARγ2 or pEGFP-N1-PPARγ2 in mediation of liposome. The former green fluorescence protein was better than the latter by fluorescence microscope. The recombinant plasmids were digested and identified. Western blot analysis showed the expression of EGFP - PPARγ2 in vitro. EGFP-PPARγ2 protein was detectable in the nucleus of BMSC. Conclusion The recombinant adenovirus encoding EGFP-PPARγ2 fusion protein was successfully constructed, which provided a basis for application of EGFP -PPARγ2 gene to adenovirus -mediated gene therapy.