人PPARγ基因过表达重组腺病毒载体的构建及鉴定  

Construction and Identification of Recombinant Adenovirus Vector Expressing Human PPARγ

作　　者：李腾[1] 李琼[1] 严国强[1] 储佳佳 沈小丹[1] 温见炳 黄起壬[1] 钟荣梅[2] 

机构地区：[1]南昌大学药学院药理学教研室江西省基础药理学重点实验室 [2]南昌大学期刊社,南昌330006

出　　处：《南昌大学学报（医学版）》2015年第3期19-22,26,共5页Journal of Nanchang University：Medical Sciences

基　　金：国家自然科学基金(81360060;81070633);江西省主要学科学术和技术带头人培养计划项目(20123BCB22005)

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。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.

关 键 词：过氧化物酶体增殖物激活受体 重组腺病毒载体 基因重组 AdMax 腺病毒包装系统 

分 类 号：Q754[生物学—分子生物学]