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

目的构建小鼠增强型绿色荧光蛋白（EGFP）-过氧化物酶体增长因子活化受体（PPAR）γ2基因的腺病毒重组体，并检测其在感染病毒的小鼠骨髓间充质干细胞（BMSC）中的表达。方法从pcDNA flag PPARγ质粒上切取目的片段PPARγ2，克隆入pEGFP-C1和pEGFP-N1，以pEGFP-C1-PPARγ2为模板通过PCR技术获得EGFP-PPARγ2基因，酶切DC315质粒，获得DC315-EGFP-PPARγ2质粒，在脂质体介导下与腺病毒辅助大质粒pBHGlox△E1、3Cre共转染HEK293细胞，包装产生复制缺陷型重组腺病毒Ad-EGFP-PPARγ2，酶切鉴定证实构建成功。转染HEK293细胞并检测其体外表达情况，经HEK293细胞扩增后，测定病毒滴度，转染小鼠BMSC，72 h后鉴定其成脂分化情况。结果将pEGFP-C1-PPARγ2和pEGFP-N1-PPARγ2通过脂质体转染入HEK293细胞后，在倒置相差显微镜下观察，前者在细胞核内有较强的绿色荧光，后者的荧光强度则极低。对重组腺病毒进行酶切和PCR鉴定，证实含小鼠EGFP-PPARγ2的Ad5腺病毒载体构建成功。在倒置相差显微镜下观察到BMSC细胞核内有绿色荧光。结论成功构建含小鼠EGFP-PPARγ2的重组Ad5腺病毒载体，为基因辅助的脂肪组织工程技术、抗肿瘤研究等提供了安全有效的转基因载体。

Construction of recombinant gene adenovirus encoding enhanced green fluorecence protein -peroxisome proliferator -activated receptor γ2 fusion protein and its expression in bone marrow mesenchymal stem cells

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.