人 PPARγ基因过表达重组腺病毒载体的构建及鉴定

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 PPARγ cDNA 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 loxΔE1，3 Cre）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 HU-VECs 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。%ABSTRACT:Objective To construct a recombinant adenovirus vector expressing human peroxi-some proliferator-activated receptor γ(PPARγ).Methods PPARγ cDNA from human umbilical vein endothelial cells (HUVECs)was digested with AgeⅠ and NheⅠ and inserted into the plas-mid CMV-MCS-SV40-EGFP to generate a recombinant shuttle plasmid.Then the recombinant shuttle plasmid and framework plasmid pBHG lox ΔE1,3 Cre were recombined with Cre/loxP system to produce the adenovirus vector expressing PPARγ (Ad-PPARγ).The Ad-PPARγ was transfected into HEK293T cells for package,amplification,purification and titer determination. Results The PCR and sequence analysis showed that the Ad-PPARγwas constructed successful-ly.Western blot analysis confirmed that the transfection with Ad-PPARγ significantly up-regula-ted the expression of PPARγ in HUVECs.Conclusion The Ad-PPARγ was successfully con-structed and markedly up-regulated the expression of PPARγin HUVECs.