应用Ad Easy载体系统构建人β_2-AR基因重组腺病毒  

Construction and Expression of the Recombinant Adenovirus Carrying Human β_2-Adrenergic Receptor

作　　者：宫海滨[1] 魏翠[1] 张清林[1] 吕茜[1] 

机构地区：[1]徐州市心血管病研究所,徐州市中心医院,江苏徐州221009

出　　处：《现代生物医学进展》2009年第4期618-621,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金资助项目(30572073);江苏省自然科学基金项目(DK2005428);江苏省卫生厅重大项目(K200402);江苏省医学重点人才(RC2007024)

摘　　要：目的:利用Ad easy腺病毒表达系统构建含人β2-肾上腺素能受体(β2-AR)基因重组腺病毒,并在HEK293细胞中扩增制备重组腺病毒。方法:将β2-AR全长cDNA插入到腺病毒穿梭载体pAdTrack-CMV,构建pAdTrackCMV-β2AR重组质粒,经Pme I酶切线性化后经电击法转入含Ad easy质粒的电感受态菌BJ5183进行重组。挑选同源重组质粒,Pac I酶切线性化转染HEK293细胞包装成重组腺病毒颗粒。将重组病毒和SD大鼠心肌细胞共培养,western-blot检测β2-AR的表达。结果:琼脂糖凝胶电泳显示在1242bp处有特异性条带后被克隆至腺病毒穿梭质粒。重组穿梭载体经Hind III和Xba I限制性酶切可以得到目的条带并经测序证实。电击法转化BJ5183所得候选重组子经PacI酶切后得到30Kb腺病毒基因组片段和4.5Kb氨苄抗性片段,证实获得同源重组质粒。转染293细胞可以看到绿色荧光蛋白,以MOI100转染SD大鼠心肌细胞发现携带β2-AR的腺病毒可以在心肌细胞中过表达。结论:利用新型腺病毒载体在短时间内成功构建了携带有人β2-AR基因的腺病毒,为进一步研究人β2-AR基因治疗奠定了基础。Objective： To construct recombinant adenovirus vector bearing the human β2-adrenergic receptor （β2-AR） and amplify the adenovirus in 293 cells. Methods： Human β2-AR cDNA（1242bp） was cloned into adenovirus shuttle plasmid pAdtrackCMV to generate a recombinant plasmid pAdtrackCMV-β2AR, then the linearized plasmid was transformed into E. coli B J5183 cells which had been electroporated adenovirus backbone plasmid pAdEasy-1. The identified recombinant plasmid was digested with Pac Ⅰ and transfected into 293 cells to package the adenovirus. Then, the SD rat cardiomyocytes co-cultured with recombinant adenovirus were used to study the expression of β2-adrenergic receptor. Results： The specific fragment （1242bp） of β2-AR gene was detected by agarose gel electrophoresis. Then the fragment was cloned into pAdtrackCMV plasmid and the recombinant plasmid pAdTrackCMV-β2AR was validated by digestion with HindlII and XbaI and sequencing. Candidate recornbinants was digested by Pac Ⅰ, which could be released into a 30kb fragment and a smaller fragment of 4.5kb, then the recombinant plasmid was gained. Green fluorescence protein expression in 293 cells was detected under fluorescent microscope after transfection. Co-cultured recombinant adenovirus （MOI100） and SD rat cardiomyoeytes, expression level of β2-AR was detected by western-blot. Conclusion： The recombinant plasmid Ad-β2AR was established rapidly and simply by homologous recombination. It may be a new tool for gene therapy using β2-AR.

关 键 词：AD Easy腺病毒 人β2-肾上腺素能受体 大鼠 心肌细胞 

分 类 号：Q784[生物学—分子生物学]