【摘要】 目的构建人的过氧化物酶体增殖物激活受体(PPARγ)基因的重组腺病毒载体。方法从人脐静脉内皮细胞(HUVECs)中提取并扩增人PPARγcDNA目的片段,将该目的片段经AgeⅠ/NheⅠ酶切后插入经相同内切酶酶切后表达质粒(CMV-MCS-SV40-EGFP)得到重组穿梭质粒;再将该重组穿梭质粒与AdMax腺病毒包装系统的骨架质粒(pBHG loxΔE1,3Cre)经Cre/loxP酶切重组,构建重组腺病毒(Ad-PPARγ);最后,将Ad-PPARγ感染HEK293T细胞,进行病毒的包装、扩增、纯化及滴度检测。结果通过PCR鉴定及基因测序分析,PPARγ过表达重组腺病毒载体构建成功;Western blot结果显示经包装及纯化的Ad-PPARγ感染HUVECs后,可显著上调HUVECs中PPARγ基因的表达。结论PPARγ过表达重组腺病毒载体构建成功,且能有效上调HUVECs中PPARγ基因的表达。更多还原

【Abstract】 Objective To construct a recombinant adenovirus vector expressing human peroxisome proliferator-activated receptorγ(PPARγ).Methods PPARγcDNA from human umbilical vein endothelial cells(HUVECs)was digested with AgeⅠ and NheⅠ and inserted into the plasmid CMV-MCS-SV40-EGFP to generate a recombinant shuttle plasmid.Then the recombinant shuttle plasmid and framework plasmid pBHG loxΔE1,3Cre were recombined with Cre/loxP system to produce the adenovirus vector expressing PPARγ(Ad-PPARγ).The Ad-PPARγwas transfected into HEK293 Tcells for package,amplification,purification and titer determination.Results The PCR and sequence analysis showed that the Ad-PPARγwas constructed successfully.Western blot analysis confirmed that the transfection with Ad-PPARγsignificantly up-regulated the expression of PPARγin HUVECs.Conclusion The Ad-PPARγ was successfully constructed and markedly up-regulated the expression of PPARγin HUVECs.更多还原